Coagulation factor ix compositions and methods of making and using same

ABSTRACT

The present invention relates to compositions comprising factor IX coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of coagulation factor-related diseases, disorders, and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/427,763, filed Feb. 8, 2017, which is a division of U.S. patentapplication Ser. No. 15/163,603, filed May 24, 2016, now U.S. Pat. No.9,758,776, which is a division of U.S. patent application Ser. No.14/710,077, filed May 12, 2015, now U.S. Pat. No. 9,376,672, which is adivision of U.S. patent application Ser. No. 13/392,511, filed Jun. 27,2012, now U.S. Pat. No. 9,062,299, which is the U.S. National Phaseapplication of International Application No. PCT/US2010/002148 filedAug. 2, 2010, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/236,493 filed Aug. 24, 2009, 61/236,836 filed Aug. 25, 2009,61/280,955, filed Nov. 10, 2009, and 61/280,956 filed Nov. 10, 2009. Allof the above listed applications are incorporated herein by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 15, 2021, isnamed 714013_5 A9-407PCUSDV4_ST25.txt, and is 1,578,544 bytes in size.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under SBIR grant2R44GM079873-02 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In hemophilia, the clotting of blood is disturbed by a lack of certainplasma blood clotting factors. Human factor IX (FIX) is a zymogen of aserine protease that is an important component of the intrinsic pathwayof the blood coagulation cascade. In individuals who do not have FIXdeficiency, the average half-life of FIX is short, approximately 18-24hours. A deficiency of functional FIX, due to an X-linked disorder thatoccurs in about one in 30,000 males, results in hemophilia B, also knownas Christmas disease, named after a young boy named Stephen Christmaswho was found to be lacking this factor. Over 100 mutations of factor IXhave been described; some cause no symptoms, but many lead to asignificant bleeding disorder. When untreated, hemophilia B isassociated with uncontrolled bleeding into muscles, joints, and bodycavities following injury, and may result in death. Previously,treatments for the disease included administration of FIX prepared fromhuman plasma derived from donor pools, which carried attendant risks ofinfection with blood-borne viruses including human immunodeficiencyvirus (HIV) and hepatitis C virus (HCV). More recently, recombinant FIXproducts have become commercially available.

The in vivo activity of exogenously supplied factor IX is limited bothby protein half-life and inhibitors of coagulation, includingantithrombin III. Factor IX compositions typically have shorthalf-lives, requiring frequent injections. Also, current FIX-basedtherapeutics requires intravenous administration due to poorbioavailability. Thus, there is a need for improved factor IXcompositions with extended half-life and retention of activity whenadministered as part of a preventive and/or therapeutic regimen forhemophilia, including hemophilia B.

Factor VII is a coagulation factor protein synthesized in the liver andsecreted into the blood as a single chain zymogen with a molecularweight of approximately 50 kDa. The FVII zymogen is converted into anactivated form (FVIIa) by proteolytic cleavage, and the activated form,when complexed with tissue factor (TF), is able to convert both factorIX and factor X into their activated forms, leading to rapid thrombingeneration and fibrin formation. Because the circulating half-life ofrFVIIa is about 2.3 hours (“Summary Basis for Approval for NovoSeven©”,FDA reference number 96-0597), multiple and frequent administrations arerequired for the treatment of bleeding disorders in hemophiliacs andsubjects with factor VII deficiency.

Chemical modifications to a therapeutic protein can reduce its in vivoclearance rate and subsequent increase serum half-life. One example of acommon modification is the addition of a polyethylene glycol (PEG)moiety, typically coupled to the protein via an aldehyde orN-hydroxysuccinimide (NHS) group on the PEG reacting with an amine group(e.g. lysine side chain or the N-terminus). However, the conjugationstep can result in the formation of heterogeneous product mixtures thatneed to be separated, leading to significant product loss and complexityof manufacturing and does not result in a completely chemically-uniformproduct. Also, the pharmacologic function of the therapetuics proteinmay be hampered if amino acid side chains in the vicinity of its bindingsite are modified by the PEGylation process. Fusing an Fc domain to thetherapeutic protein is another approach to increases the size of thetherapeutic protein, hence reducing the rate of clearance through thekidney. Additionally, the Fc domain confers the ability to bind to, andbe recycled from lysosomes by, the FcRn receptor, which results inincreased pharmacokinetic half-life. Unfortunately, the Fc domain doesnot fold efficiently during recombinant expression, and tends to forminsoluble precipitates known as inclusion bodies. These inclusion bodiesmust be solubilized and functional protein must be renatured from themisfolded aggregate. Such process is time-consuming, inefficient, andexpensive. Accordingly, there remains a need for improved coagulationfactor compositions with increased half-life which can be administeredless frequently, and/or be produced by a simpler process at a cheapercost.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods for thetreatment or improvement of a condition or the enhancement of aparameter associated with the administration of coagulations factors IXand/or VII. In particular, the present invention provides compositionsof fusion proteins comprising one or more extended recombinantpolypeptides (XTEN). A subject XTEN is typically a non-repetitivesequence and unstructured conformation. XTEN is linked to a coagulationfactor (“CF”) selected from factor IX (“FIX”), factor VII (“FVII”),factor VII-factor IX hybrids, and sequence variants thereof, resultingin a coagulation factor-XTEN fusion protein (“CFXTEN”). In part, thepresent disclosure is directed to pharmaceutical compositions comprisingthe fusion proteins and the uses thereof for treating coagulationfactor-related diseases, disorders or conditions. The CFXTENcompositions have enhanced pharmacokinetic properties compared to CF notlinked to XTEN, which may permit more convenient dosing and improvedefficacy. In some embodiments, the CFXTEN compositions of the inventiondo not have a component selected the group consisting of: polyethyleneglycol (PEG), albumin, antibody, and an antibody fragment.

In some embodiments, the invention provides an isolated factor IX fusionprotein, comprising a factor IX sequence that is at least about 90%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%identical to an amino acid sequence selected from Table 1. The factor IXhaving such sequence identity is further linked to an extendedrecombinant polypeptide (XTEN) having at least about 100 to about 3000amino acid residues. In one embodiment, the XTEN is linked to theC-terminus of the FIX or the FVII CF. In some embodiments, the inventionprovides an isolated factor VII fusion protein, comprising a factor VIIthat is at least about 90%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% identical to an amino acid sequence selectedfrom Table 2. The factor VII having such sequence is linked to anextended recombinant polypeptide (XTEN).

Non-limiting examples of CFXTEN with a single FIX or a single FVIIlinked to a single XTEN are presented in Table 41. In one embodiment,the invention provides a CFXTEN composition has at least about 80%sequence identity compared to a CFXTEN from Table 41, alternatively atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity ascompared to a CFXTEN from Table 41. In some embodiments, the CF and theXTEN components of the fusion protein are linked via a cleavage sequencethat is cleavable by a protease, including endogenous mammalianproteases. Examples of such protease include, but are not limited to,FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, thrombin, elastase-2,granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, TEV, enterokinase,rhinovirus 3C protease, and sortase A, or a sequence selected from Table7. In one embodiment, a CFXTEN composition with a cleavage sequence hasa sequence having at least about 80% sequence identity compared to aCFXTEN from Table 42, alternatively at least about 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or about 100% sequence identity as compared to a CFXTEN from Table42. However, the invention also provides substitution of any of the CFsequences of Table 1 or Table 2 for a CF in a sequence of Table 42, andsubstitution of any XTEN sequence of Table 4 for an XTEN in a sequenceof Table 42, and substitution of any cleavage sequence of Table 7 for acleavage sequence in a sequence of Table 42. In CFXTEN embodimentshaving cleavage sequences, cleavage of the cleavage sequence by theprotease releases the XTEN from the CF. In some embodiments of theforegoing, the CF component becomes biologically active or has anincrease in activity upon its release from the XTEN by cleavage of thecleavage sequence, wherein the pro-coagulant activity is at least about30%, or at least about 40%, or at least about 50%, or at least about60%, or at least about 70%, or at least about 80%, or at least about 90%compared to the corresponding FIX or FVII not linked to XTEN.

The invention provides isolated CFXTEN fusion proteins that comprise asecond XTEN of about 36 to about 3000 amino acid residues, which can beidentical or can be different from the first XTEN, wherein the secondXTEN can be incorporated between any two adjacent domains of the CF,i.e., between the Gla, EFG1, EGF2, activating peptide and proteasedomains, or is incorporated within the sequence of an existing loopdomain of a domain sequence of the CF, as described more fully in theExamples. In one embodiment, the first and the second XTEN can be anamino acid sequence selected from any one of Tables 4, or 9-13, or canexhibit at least at least about 80%, or at least about 90%, or at leastabout 91%, or at least about 92%, or at least about 93%, or at leastabout 94%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% sequenceidentity compared to a sequence selected from Tables 4 and 9-13. Inanother embodiment, the isolated fusion protein comprises a second XTENof about 36 to about 3000 amino acid residues. The fusion protein canadopt a multiple-XTEN configuration of Table 6, or a variation thereof.

The invention provides CFXTEN compositions comprising XTEN linked to afactor VII comprising one or more heterologous cleavage sequencescleavable by the same or different pro-coagulant proteases. In someembodiments of the foregoing, the factor VII comprises a heterologoussequence of factor IX incorporated into or substituted for portions ofthe FVII sequence, resulting in factor VII-factor IX hybrid sequencevariants. In some embodiments, a portion or the entirety of the sequencefrom the activation peptide domain of FIX is incorporated or substitutedfor FVII sequences bridging the region between the EFG2 and proteasedomains of the FVII component, resulting in compositions that can beactivated as part of the intrinsic system of the coagulation cascade(e.g., activated factor XI). In such case, the factor VII-factor IXCFXTEN composition can be activated by a pro-coagulant protease in theabsence of tissue factor, such that the CFXTEN can serve as a by-pass offactors VIII and IX in the intrinsic coagulation pathway when suchfactors are deficient (e.g., in hemophilia A or B) or when inhibitors tothese factors are present. In one embodiment, the FVII-FIX sequencevariant incorporates the full-length FIX AP domain plus at least about2, or at least about 3, or at least about 4, or at least about 5, or atleast about 6, or at least about 7, or at least about 8, or at leastabout 9, or at least about 10, or at least about 11, or at least about12 or more amino acids flanking adjacent amino acid residues on one orboth sides of the R145-A146 and R180-V181 cleavage sites of the FIX APdomain (e.g., the sequenceRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE (SEQ ID NO: 1) inthe case of 12 flanking amino acids on the N-terminus side and 5flanking amino acids on the C-terminus side).

In another embodiment, the CFXTEN FVII-FIX sequence variant comprises aheterologous FIX sequence exhibiting at least at least about 80%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or 100%identity compared to the sequence:KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV (SEQ ID NO: 2), when optimallyaligned.

In other embodiments, the CFXTEN comprises FVII-FIX sequence variantsthat incorporate a portion of the FIX AP that includes a sequence of atleast about 2, or at least about 3, or at least about 4, or at leastabout 5, or more amino acids that flank one or both sides of theR145-A146 cleavage site (e.g., the sequence TSKLTRAETVFP (SEQ ID NO: 3)in the case of 6 flanking amino acids on either side of the cleavagesite) or a sequence of at least about 2, or at least about 3, or atleast about 4, or at least about 5 or more amino acids that flank one orboth sides of the R180-V181 cleavage site (e.g., the sequence and DFTRV(SEQ ID NO: 4) in the case of 4 amino acids on the N-terminal flank andvaline as the C-terminus of the cleavage site from FIX). In oneembodiment of the foregoing, the CFXTEN FVII-FIX sequence variantcomprises a heterologous FIX sequence exhibiting at least at least about80%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or 100% identity compared to a sequence selected from TSKLTRAETVFP(SEQ ID NO: 3) and FNDFTRV (SEQ ID NO: 5), when optimally aligned.

In another embodiment, the CFXTEN comprises a FVII-FIX sequence variantdisclosed above that further includes the same AP cleavage sequence as alinker between the C-terminus of the FVII component and the XTENcomponent of the fusion protein, e.g., an N- to C-terminus configurationof FVII variant-AP sequence-XTEN, thereby permitting the release of theFVII variant component from the CFXTEN fusion protein when cleaved bythe same pro-coagulant protease as per that of the FVII to FVIIatransition. In one embodiment, the FVII-FIX CFXTEN of any of theforegoing embodiments includes the factor XI cleavage sequence KLTRAET(SEQ ID NO: 6) as the linker between the FVII-FIX sequence and the XTEN,thereby permitting the release of the FVII variant component from theCFXTEN fusion protein by the initiation of the intrinsic coagulationcascade. In one embodiment, the invention provides a CFXTEN with aFVII-FIX hybrid sequence that exhibits at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98%, or at leastabout 99%, sequence identity compared to a sequence from Table 43. Inother embodiments, the invention provides a FVII-FIX sequence variantwith incorporated FIX-derived AP cleavage sequence that is not linked toan XTEN. In one embodiment, the FVII-FIX sequence without an XTENexhibits at least about 80%, or at least about 85%, or at least about90%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity ascompared with a sequence from Table 43 without an XTEN.

In one embodiment of the CFXTEN composition, the invention provides afusion protein of formula I:

(XTEN)_(x)—CF-(XTEN)_(y)  I

wherein independently for each occurrence, CF is a coagulation factor; xis either 0 or 1 and y is either 0 or 1 wherein x+y≥1; and XTEN is anextended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesa fusion protein of formula II:

(XTEN)_(x)-(CF)-(S)_(y)-(XTEN)_(y)  II

wherein independently for each occurrence, CF is a coagulation factor a;S is a spacer sequence having between 1 to about 50 amino acid residuesthat can optionally include a cleavage sequence; x is either 0 or 1 andy is either 0 or 1 wherein x+y≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein, wherein the fusion protein is of formulaIII:

(XTEN)_(x)-(S)_(x)-(CF)-(S)_(y)-(XTEN)_(y)  III

wherein independently for each occurrence, CF is a coagulation factor; Sis a spacer sequence having between 1 to about 50 amino acid residuesthat can optionally include a cleavage sequence; x is either 0 or 1 andy is either 0 or 1 wherein x+y≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula IV:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(XTEN)_(w)-(AP)-(XTEN)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  IV

wherein independently for each occurrence, Gla is a Gla domain of FIX;EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP is anactivator peptide of FIX; PRO is a protease domain of FIX; S is a spacersequence having between 1 to about 50 amino acid residues that canoptionally include a cleavage sequence; u is either 0 or 1; v is either0 or 1; x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 withthe proviso that u+v+w+x+z≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula V:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(AP1)-(XTEN)_(w)-(AP2)-(XTEN)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  V

wherein independently for each occurrence, Gla is a Gla domain of FIX;EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP1 is theN-terminal sequence portion of the activator peptide domain of FIX thatincludes a first native cleavage sequence of the AP domain; AP2 is theC-terminal sequence portion of the activator peptide domain of FIX thatincludes a second native cleavage sequence of the AP domain; PRO is aprotease domain of FIX; S is a spacer sequence having between 1 to about50 amino acid residues that can optionally include a cleavage sequence;u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+z≥1; and XTENis an extended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula VI:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(XTEN)_(w)-(Pro)-(S)_(x)-(XTEN)_(y)  VI

wherein independently for each occurrence, Gla is a Gla domain of FVII;EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is aprotease domain of FVII; S is a spacer sequence having between 1 toabout 50 amino acid residues that can optionally include a cleavagesequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; yis either 0 or 1 with the proviso that u+v+w+y≥1; and XTEN is anextended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula VII:

(Gla)-(XTEN)_(t)-(EGF1)-(XTEN)_(u)-(EGF2)-(AP1)_(v)-(XTEN)_(w)-(AP2)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  VII

wherein independently for each occurrence, Gla is a Gla domain of FVII;EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is aprotease domain of FVII; AP1 is the N-terminal sequence portion of theactivator peptide domain of FIX that includes the native cleavagesequence; AP2 is the C-terminal sequence portion of the activatorpeptide domain of FIX that includes the native cleavage sequence; S is aspacer sequence having between 1 to about 50 amino acid residues thatcan optionally include a cleavage sequence; t is either 0 or 1; u iseither 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or1; z is either 0 or 1 with the proviso that t+u+w+z≥1; and XTEN is anextended recombinant polypeptide. In the embodiment, the CFXTENcomposition can include the entirety of the FIX activator peptide domainsequence or one or both cleavage sequences from the activator peptidedomain of factor IX, e.g., a sequence of at least about 3 to about 12amino acids that flank the R145-A146 cleavage site and the sequence ofat least about 1 to about 5 amino acids that flank the R180-V181cleavage site, as described more fully above. The invention alsocontemplates substitution of any of the other cleavage sequences ofTable 7 for the AP cleavage sequences.

The CFXTEN compositions of the embodiments described herein can beevaluated for retention of activity (including after cleavage of anyincorporated XTEN-releasing cleavage sites) using any appropriate invitro assay disclosed herein (e.g., the assays of Table 40 or the assaysdescribed in the Examples), to determine the suitability of theconfiguration for use as a therapeutic agent in the treatment of acoagulation-factor related disease, disorder or condition. In oneembodiment, the CFXTEN exhibits at least about 60%, or at least about70%, or at least about 80%, or at least about 90% of the activitycompared to the native CF not linked to XTEN. In another embodiment, theCF component released from the CFXTEN by enzymatic cleavage of theincorporated cleavage sequence linking the CF and XTEN componentsexhibits at least about 60%, or at least about 70%, or at least about80%, or at least about 90% of the activity compared to the native CF notlinked to XTEN.

The XTEN of the CFXTEN compositions have at least about 200, or at leastabout 400, or at least about 800, or at least about 900, or at leastabout 1000, or at least about 2000, up to about 3000 amino acidsresidues. The XTEN of the CFXTEN fusion protein compositions ischaracterized in that they have one or more of the followingcharacteristics: (a) at least a first XTEN comprises at least about 200contiguous amino acids that exhibits at least about 90%, or about 95%,or about 96%, or about 97%, or about 98%, or about 99% identity to acomparable length of an amino acid sequence selected from a sequenceshown in Table 4; (b) the XTEN sequence lacks a predicted T-cell epitopewhen analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithmprediction for epitopes within the XTEN sequence is based on a score of−5, or −6, or −7, or −8, or −9 or greater; (c) the XTEN has asubsequence score of less than 10, or less than 9, or less than 8, orless than 7, or less than 6, or less than 5, or even less; (d) the sumof asparagine and glutamine residues is less than 10% of the total aminoacid sequence of the XTEN; (e) the sum of methionine and tryptophanresidues is less than 2% of the total amino acid sequence of the XTEN;(f) the XTEN has greater than 90% random coil formation, or about 95%,or about 96%, or about 97%, or about 98%, or about 99% random coilformation as determined by GOR algorithm; (g) the XTEN sequence has lessthan 2% alpha helices and 2% beta-sheets as determined by theChou-Fasman algorithm; and (h) the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P) residuesconstitutes more than about 90%, or about 95%, or about 96%, or about97%, or about 98%, or about 99% of the total amino acid residues of theXTEN.

In another embodiment, the invention provides CFXTEN fusion proteins,wherein the XTEN is characterized in that the sum of asparagine andglutamine residues is less than 10% of the total amino acid sequence ofthe XTEN, the sum of methionine and tryptophan residues is less than 2%of the total amino acid sequence of the XTEN, the XTEN sequence has lessthan 5% amino acid residues with a positive charge, the XTEN sequencehas greater than 90% random coil formation, or about 95%, or about 96%,or about 97%, or about 98%, or about 99% random coil formation asdetermined by GOR algorithm; and the XTEN sequence has less than 2%alpha helices and 2% beta-sheets as determined by the Chou-Fasmanalgorithm. In some embodiments, no one type of amino acid constitutesmore than 30% of the XTEN sequence of the CFXTEN.

In another embodiment, the invention provides CFXTEN fusion proteins,wherein the XTEN is characterized in that at least about 80%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% of the XTEN sequence consists of non-overlappingsequence motifs wherein each of the sequence motifs has about 9 to about14 amino acid residues and wherein the sequence of any two contiguousamino acid residues does not occur more than twice in each of thesequence motifs consist of four to six types of amino acids selectedfrom glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)and proline (P). In one embodiment, the XTEN is characterized in that atleast about 80%, or at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% of the XTEN sequence consists ofnon-overlapping sequence motifs wherein the motifs are selected fromTable 3.

In some embodiments, the XTEN has a sequence in which no threecontiguous amino acids are identical unless the amino acid is serine, inwhich case no more than three contiguous amino acids are serineresidues. In other embodiment, the XTEN component of the CFXTEN has asubsequence score of less than 10, or less than 9, or less than 8, orless than 7, or less than 6, or less than 5, or less. In the embodimentsof this paragraph, the XTEN is characterized as “substantiallynon-repetitive.”

In some embodiments, the invention provides CFXTEN comprising at least asecond XTEN, wherein the XTEN sequence exhibits at least about 80%, orat least about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% sequence identity compared to a sequence from Table 4,Table 9, Table 10, Table 11, Table 12, or Table 13.

In some embodiments, CFXTEN fusion proteins exhibits enhancedpharmacokinetic properties compared to CF not linked to XTEN, whereinthe enhanced properties include but are not limited to longer terminalhalf-life, larger area under the curve, increased time in which theblood concentration remains within the therapeutic window, increasedtime between consecutive doses results in blood concentrations withinthe therapeutic window, and decreased dose in moles over time that canbe administered compared to a CF not linked to XTEN, yet still result ina blood concentration within the therapeutic window for thatcomposition. In some embodiments, the terminal half-life of the CFXTENfusion protein administered to a subject is increased at least aboutthree-fold, or at least about four-fold, or at least about five-fold, orat least about six-fold, or at least about eight-fold, or at least aboutten-fold, or at least about 20-fold, or at least about 40-fold, or atleast about 60-fold, or at least about 100-fold, or even higher ascompared to CF not linked to XTEN and administered to a subject at acomparable dose. In other embodiments, the terminal half-life of theCFXTEN fusion protein administered to a subject is at least about 12 h,or at least about 24 h, or at least about 48 h, or at least about 72 h,or at least about 96 h, or at least about 120 h, or at least about 144h, or at least about 21 days or greater. In other embodiments, theenhanced pharmacokinetic property is reflected by the fact that theblood concentrations that remain within the therapeutic window for theCFXTEN fusion protein for a given period are at least about two fold, orat least about three-fold, or at least about four-fold, or at leastabout five-fold, or at least about six-fold, or at least abouteight-fold, or at least about ten-fold longer, or at least about20-fold, or at least about 40-fold, or at least about 60-fold, or atleast about 100-fold compared to CF not linked to XTEN and administeredto a subject at a comparable dose. The increase in half-life and timespent within the therapeutic window permits less frequent dosing anddecreased amounts of the fusion protein (in moles equivalent) that areadministered to a subject, compared to the corresponding CF not linkedto XTEN. In one embodiment, administration of a CFXTEN to a subjectusing a therapeutically-effective dose regimen results in a gain in timeof at least two-fold, or at least three-fold, or at least four-fold, orat least five-fold, or at least six-fold, or at least eight-fold, or atleast 10-fold, or at least about 20-fold, or at least about 40-fold, orat least about 60-fold, or at least about 100-fold or higher between atleast two consecutive C_(max) peaks and/or C_(min) troughs for bloodlevels of the fusion protein compared to the corresponding CF not linkedto the XTEN and administered using a comparable dose regimen to asubject.

In some embodiments, the XTEN enhances thermostability of CF when linkedto the XTEN wherein the thermostability is ascertained by measuring theretention of biological activity after exposure to a temperature ofabout 37° C. for at least about 7 days of the biologically activeprotein in comparison to the biologically active protein not linked tothe XTEN. In one embodiment of the foregoing, the retention ofbiological activity increases by at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 100%, or about 150%, at least about 200%, at least about 300%, orabout 500% longer compared to the CF not linked to the XTEN.

In some embodiments, the isolated CFXTEN fusion protein is configures tohave reduced binding affinity for a clearance receptor as compared tothe corresponding CF not linked to the XTEN. In one embodiment, theCFXTEN fusion protein exhibits binding affinity for a clearance receptorof the CF in the range of about 0.01%-30%, or about 0.1% to about 20%,or about 1% to about 15%, or about 2% to about 10% of the bindingaffinity of the corresponding CF not linked to the XTEN. In anotherembodiment, a CFXTEN fusion protein with reduced affinity can havereduced active clearance and a corresponding increase in half-life of atleast about 3-fold, or at least about 5-fold, or at least about 6-fold,or at least about 7-fold, or at least about 8-fold, or at least about9-fold, or at least about 10-fold, or at least about 12-fold, or atleast about 15-fold, or at least about 17-fold, or at least about20-fold, or at least about 30-fold, or at least about 50-fold, or atleast about 100-fold longer compared to the corresponding CF that is notlinked to the XTEN.

In some embodiments, the invention provides CFXTEN fusion proteinswherein the CFXTEN exhibits increased solubility of at least three-fold,or at least about four-fold, or at least about five-fold, or at leastabout six-fold, or at least about seven-fold, or at least abouteight-fold, or at least about nine-fold, or at least about ten-fold, orat least about 15-fold, or at least a 20-fold, or at least 40-fold, orat least 60-fold at physiologic conditions compared to the CF not linkedto XTEN.

In some embodiments, CFXTEN fusion proteins exhibit an increasedapparent molecular weight as determined by size exclusionchromatography, compared to the actual molecular weight. In someembodiments the CF comprising a FIX and at least a first XTEN exhibitsan apparent molecular weight of at least about 400 kD, or at least about500 kD, or at least about 700 kD, or at least about 1000 kD, or at leastabout 1400 kD, or at least about 1600 kD, or at least about 1800 kD, orat least about 2000 kD, while the actual molecular weight of each FIXcomponent of the fusion protein is about 50 kD and the molecular weightof the fusion protein ranges from about 70 to about 125 kDa. In otherembodiments, the CF comprising a FVII and at least a first XTEN exhibitsan apparent molecular weight of at least about 400 kD, or at least about500 kD, or at least about 700 kD, or at least about 1000 kD, or at leastabout 1400 kD, or at least about 1600 kD, or at least about 1800 kD, orat least about 2000 kD, while the actual molecular weight of each FIXcomponent of the fusion protein is about 50 kD and the molecular weightof the fusion protein ranges from about 70 to about 125 kDa.Accordingly, the CFXTEN fusion proteins can have an apparent molecularweight that is about 6-fold greater, or about 8-fold greater, or about10-fold greater, or about 12-fold greater, or about 15-fold greater thanthe actual molecular weight of the fusion protein. In some cases, theisolated CFXTEN fusion protein of any of the embodiments disclosedherein exhibit an apparent molecular weight factor under physiologicconditions that is greater than about 4, or about 5, or about 6, orabout 7, or about 8, or about 10, or greater than about 15.

In some embodiments, administration of a therapeutically effective doseof a fusion protein of one of formulae I-VII to a subject in needthereof can result in a gain in time of at least two-fold, or at leastthree-fold, or at least four-fold, or at least five-fold or more spentwithin a therapeutic window for the fusion protein compared to thecorresponding CF not linked to the XTEN of and administered at acomparable dose to a subject. In other cases, administration of atherapeutically effective dose of a fusion protein of an embodiment offormulas I-VII to a subject in need thereof can result in a gain in timebetween consecutive doses necessary to maintain a therapeuticallyeffective dose regimen of at least 48 h, or at least 72 h, or at leastabout 96 h, or at least about 120 h, or at least about 7 days, or atleast about 14 days, or at least about 21 days between consecutive dosescompared to a CF not linked to XTEN and administered at a comparabledose.

The fusion proteins of the disclosed compositions can be designed tohave different configurations, N- to C-terminus, of a CF and XTEN andoptional spacer sequences, including but not limited to XTEN-CF,CF-XTEN, XTEN-S-CF, CF-S-XTEN, XTEN-CF-XTEN, CF-CF-XTEN, XTEN-CF-CF,CF-S-CF-XTEN, XTEN-CF-S-CF, and multimers thereof. The choice ofconfiguration can, as disclosed herein, confer particularpharmacokinetic, physico/chemical, or pharmacologic propertiesincluding, in the case of an incorporated cleavage sequence, the releaseof the CF with a concomitant increase in activity.

In some embodiments, the CFXTEN fusion protein is characterized in that:(i) it has a longer half-life when administered to a subject compared tothe corresponding coagulation factor not linked to the XTEN administeredto a subject under an otherwise equivalent dose; (ii) when a smallermolar amount of the fusion protein is administered to a subject incomparison to the corresponding coagulation factor that lacks the XTENadministered to a subject under an otherwise equivalent dose regimen,the fusion protein achieves a comparable area under the curve (AUC) asthe corresponding coagulation factor not linked to the XTEN; (iii) whena smaller molar amount of the fusion protein is administered to asubject in comparison to the corresponding coagulation factor that lacksthe XTEN administered to a subject under an otherwise equivalent doseregimen, the fusion protein achieves a comparable therapeutic effect asthe corresponding coagulation factor not linked to the XTEN; (iv) whenthe fusion protein is administered to a subject less frequently incomparison to the corresponding coagulation factor not linked to theXTEN administered to a subject using an otherwise equivalent molaramount, the fusion protein achieves a comparable area under the curve(AUC) as the corresponding coagulation factor not linked to the XTEN;(v) when the fusion protein is administered to a subject less frequentlyin comparison to the corresponding coagulation factor not linked to theXTEN administered to a subject using an otherwise equivalent molaramount, the fusion protein achieves a comparable therapeutic effect asthe corresponding coagulation factor not linked to the XTEN; (vi) whenan accumulatively smaller molar amount of the fusion protein isadministered to a subject in comparison to the corresponding coagulationfactor not linked to the XTEN administered to a subject under anotherwise equivalent dose period, the fusion protein achieves comparablearea under the curve (AUC) as the corresponding coagulation factor notlinked to the XTEN; or (vii) when an accumulatively smaller molar amountof the fusion protein is administered to a subject in comparison to thecorresponding coagulation factor not linked to the XTEN administered toa subject under an otherwise equivalent dose period, the fusion proteinachieves comparable therapeutic effect as the corresponding coagulationfactor not linked to the XTEN.

The invention provides a method of producing a fusion protein comprisinga factor VII or factor IX or a factor VII-factor IX hybrid coagulationfactor fused to one or more extended recombinant polypeptides (XTEN),comprising: (a) providing host cell comprising a recombinantpolynucleotide molecule encoding the fusion protein (b) culturing thehost cell under conditions permitting the expression of the fusionprotein; and (c) recovering the fusion protein from the culture. In oneembodiment of the method, the coagulation factor of the fusion proteinhas at least 90% sequence identity compared to a sequence selected fromTable 1 or Table 2. In another embodiment of the method, the one or moreXTEN of the expressed fusion protein has at least about 90%, or about91%, or about 92%, or about 93%, or about 94%, or about 95%, or about96%, or about 97%, or about 98%, or about 99% to about 100% sequenceidentity compared to a sequence selected from Table 4. In anotherembodiment of the method, the host cell is a eukaryotic cell. In anotherembodiment of the method, the host cell is CHO cell. In anotherembodiment of the method the isolated fusion protein is recovered fromthe host cell cytoplasm in substantially soluble form.

The invention provides isolated nucleic acids comprising apolynucleotide sequence selected from (a) a polynucleotide encoding thefusion protein of any of the foregoing embodiments, or (b) thecomplement of the polynucleotide of (a). In one embodiment, theinvention provides an isolated nucleic acid comprising a polynucleotidesequence that has at least 80% sequence identity, or about 85%, or atleast about 90%, or about 91%, or about 92%, or about 93%, or about 94%,or about 95%, or about 96%, or about 97%, or about 98%, or about 99% toabout 100% sequence identity compared to (a) a polynucleotide sequenceof comparable length selected from Table 41 and Table 42; or (b) thecomplement of the polynucleotide of (a). The invention providesexpression vectors comprising the nucleic acid of any of the embodimentshereinabove described in this paragraph. In one embodiment, theexpression vector of the foregoing further comprises a recombinantregulatory sequence operably linked to the polynucleotide sequence. Inanother embodiment, the polynucleotide sequence of the expressionvectors of the foregoing is fused in frame to a polynucleotide encodinga secretion signal sequence, which can be a CF native signal sequence.The invention provides a host cell that comprises an expression vectorof any of the embodiments hereinabove described in this paragraph. Inone embodiment, the host cell is a eukaryotic cell. In anotherembodiment, the host cell is a CHO cell. In another embodiment, the hostcell is HEK cell.

In one embodiment, the invention provides pharmaceutical compositionscomprising the fusion protein of any of the foregoing embodiments and apharmaceutically acceptable carrier. In another embodiment, theinvention provides kits, comprising packaging material and at least afirst container comprising the pharmaceutical composition of theforegoing embodiment and a label identifying the pharmaceuticalcomposition and storage and handling conditions, and a sheet ofinstructions for the reconstitution and/or administration of thepharmaceutical compositions to a subject.

The invention provides a method of treating a coagulopathy or acoagulation factor-related disease, disorder or condition in a subject,comprising administering to the subject a therapeutically effectiveamount of a CFXTEN fusion protein of any of the foregoing embodiments.In one embodiment of the method, the coagulation-factor relatedcondition is selected from bleeding disorders (e.g., defective plateletfunction, thrombocytopenia or von Willebrand's disease), coagulopathies(any disorder of blood coagulation, including coagulation factordeficiencies), hemophilia B (aka Christmas disease), factor IX-relatedbleeding disorders, factor VII deficiency, hemophilia A, vascularinjury, uncontrolled bleeding in subjects not suffering from hemophilia,bleeding from trauma or surgery, bleeding due to anticoagulant therapy,and bleeding due to liver disease. In one embodiment of the method oftreatment, the coagulopathy is hemophilia A. In one embodiment of themethod of treatment, the coagulopathy is hemophilia B. In anotherembodiment of the method of treatment, the coagulopathy is factor VIIdeficiency. In another embodiment of the method of treatment, the CFXTENis administered to a subject to control a bleeding episode. In anotherembodiment of the method of treatment, a CFXTEN comprising a factorVII-factor IX sequence hybrid is administered to a subject to control ableeding episode, wherein the CFXTEN is activated by a pro-coagulantprotease of the intrinsic coaguation cascade (e.g., activated factorXI). In another embodiment, the present invention provides a method oftreating a clotting factor deficiency in a subject, comprising:administering to said subject a composition comprising a therapeuticallyeffective amount of the factor VII provided herein.

In some embodiments, the composition can be administered subcutaneously,intramuscularly, or intravenously. In one embodiment, the composition isadministered at a therapeutically effective amount, wherein theadministration results in a gain in time spent within a therapeuticwindow for the fusion protein compared to the corresponding CF of thefusion protein not linked to the XTEN and administered at a comparabledose to a subject. The gain in time spent within the therapeutic windowcan at least three-fold longer than the corresponding CF not linked tothe XTEN, or alternatively, at least four-fold, or five-fold, orsix-fold, or seven-fold, or eight-fold, or nine-fold, or at least10-fold, or at least 20-fold, or at least about 30-fold, or at leastabout 50-fold, or at least about 100-fold longer than the correspondingCF not linked to XTEN. In some embodiments of the method of treatment,(i) a smaller molar amount of (e.g. of about two-fold less, or aboutthree-fold less, or about four-fold less, or about five-fold less, orabout six-fold less, or about eight-fold less, or about 100 fold-less orgreater) the fusion protein is administered in comparison to thecorresponding coagulation factor not linked to the XTEN under anotherwise same dose regimen, and the fusion protein achieves acomparable area under the curve and/or a comparable therapeutic effectas the corresponding coagulation factor not linked to the XTEN; (ii) thefusion protein is administered less frequently (e.g., every two days,about every seven days, about every 14 days, about every 21 days, orabout, monthly) in comparison to the corresponding coagulation factornot linked to the XTEN under an otherwise same dose amount, and thefusion protein achieves a comparable area under the curve and/or acomparable therapeutic effect as the corresponding coagulation factornot linked to the XTEN; or (iii) an accumulative smaller molar amount(e.g. about 5%, or about 10%, or about 20%, or about 40%, or about 50%,or about 60%, or about 70%, or about 80%, or about 90% less) of thefusion protein is administered in comparison to the correspondingcoagulation factor not linked to the XTEN under the otherwise same doseregimen the fusion protein achieves a comparable area under the curveand/or a comparable therapeutic effect as the corresponding coagulationfactor not linked to the XTEN. The accumulative smaller molar amount ismeasured for a period of at least about one week, or about 14 days, orabout 21 days, or about one month. In some embodiments of the method oftreatment, the therapeutic effect is a measured parameter selected fromblood concentrations of coagulation factor, prothrombin (PT) assay,activated partial prothrombin (aPTT) assay, bleeding time assay, wholeblood clotting time (WBCT), and thrombelastography.

In another embodiment, invention provides a method of treating adisease, disorder or condition, comprising administering thepharmaceutical composition described above to a subject using multipleconsecutive doses of the pharmaceutical composition administered using atherapeutically effective dose regimen. In one embodiment of theforegoing, the therapeutically effective dose regimen can result in again in time of at least three-fold, or alternatively, at leastfour-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, ornine-fold, or at least 10-fold, or at least 20-fold, or at least about30-fold, or at least about 50-fold, or at least about 100-fold longertime between at least two consecutive C_(max) peaks and/or C_(min)troughs for blood levels of the fusion protein compared to thecorresponding CF of the fusion protein not linked to the fusion proteinand administered at a comparable dose regimen to a subject. In anotherembodiment of the foregoing, the administration of the fusion proteinresults in improvement in at least one measured parameter of acoagulation factor-related disease using less frequent dosing or a lowertotal dosage in moles of the fusion protein of the pharmaceuticalcomposition compared to the corresponding biologically active proteincomponent(s) not linked to the fusion protein and administered to asubject d using a therapeutically effective regimen to a subject.

The invention further provides use of the compositions comprising thefusion protein of any of the foregoing embodiments in the preparation ofa medicament for treating a disease, disorder or condition in a subjectin need thereof. In one embodiment of the foregoing, the disease,disorder or condition is selected from group consisting of bleedingdisorders, coagulopathies, hemophilia B (aka Christmas disease), factorIX-related bleeding disorders, factor VII deficiency, vascular injury,bleeding from trauma or surgery, bleeding due to anticoagulant therapy,and liver disease. Any of the disclosed embodiments can be practicedalone or in combination depending on the interested application.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the invention may be further explained byreference to the following detailed description and accompanyingdrawings that sets forth illustrative embodiments.

FIGS. 1A and 1B show schematic representations of exemplary CFXTEN(FIX-XTEN) fusion proteins. FIG. 1A shows the domain architecture ofnative FIX, with the gamma-carboxyglutamate domain, the EGF1 and EGF2domains, the activation peptide, and the protease domain, with a linkedXTEN at the C-terminus. Arrows indicate the cleavage sites for theactivation peptide domain. FIG. 1B shows a FIX molecule with an XTENpolypeptide attached to the C-terminus via a cleavage sequence, andindicates a site for proteolytic cleavage to release the XTEN (arrowsindicate the cleavage sites for the activation peptide domain and therelease point for the XTEN).

FIGS. 2A-2F illustrate several examples of CXTEN configurations ofFIX-XTEN and associated protease cleavage sites. FIG. 2A shows anFIX-XTEN with two proteolytic cleavage sites (arrows) within theactivation peptide of FIX, and a C-terminus XTEN without a cleavage sitelinkage. FIG. 2B is similar to the configuration of FIG. 2A, but theC-terminus XTEN is linked via a cleavage sequence, with the arrowindicating the release point. FIG. 2C shows three configurations ofFIX-XTEN, with the XTEN integrated between the various domains of FIX.FIG. 2D shows an FIX-XTEN with the XTEN portion inserted into theactivation peptide between the native cleavage sites, which wouldrelease the XTEN upon the proteolytic activation of FIX. FIG. 2Eillustrates FIX-XTEN that contain multiple XTEN sequences insertedbetween different domains with the addition of a releasable XTEN at theC-terminus. FIG. 2F illustrates FIX-XTEN where the XTEN has beeninserted within loop domains of FIX.

FIG. 3 is a schematic of the coagulation cascade, showing both theextrinsic and intrinsic pathways.

FIGS. 4A-4D show several examples of CXTEN configurations of FVII-XTEN.FIG. 4A shows a FVII-XTEN that has not been activated. FIG. 4B shows aFVII-XTEN in which the peptide has been cleaved, resulting in anactivated FVIIa-XTEN; FIG. 4C illustrates a FVII-XTEN composition with acleavage sequence for releasable XTEN in which the FVII component hasnot been activated, containing a cleavage site for the activationprotease (AP) and a second cleavage site for the release protease (RP).FIG. 4D shows a composition of activated FVIIa-XTEN containing acleavage site for the release protease.

FIGS. 5A-5E illustrate a strategy for FVII-XTEN design approach usinginternal XTEN. FIGS. 5A-5D show exemplary sites for XTEN insertionbetween boundaries of the FVII domains with inactive FVII on the leftand an activated form of FVII on the right (FIG. 5A: Insertion of XTENbetween Gla and EGF1 domain, FIG. 5B: Insertion of XTEN between EGF1 andEGF2. FIG. 5C: Insertion of XTEN at C-terminus of activation peptide,FIG. 5D: Insertion of XTEN at N-terminus of activation peptide). FIG. 5Eshows examples of FVII-XTEN in which the XTEN is located within externalloops within individual domains fusion proteins, with inactive FVII onthe left and FVIIa on the right. The activation peptide in FVII is shownas a thin line versus XTEN that is shown as a fat line.

FIG. 6 illustrates essentially the same constructs as FIG. 5, but withan XTEN linked at the C-terminus of each construct.

FIG. 7 is a schematic that shows some of the various locations in whichXTEN can be inserted internal to the sequences of the coagulationfactors FVII or FIX.

FIG. 8 is a schematic of the key components of the clotting system. FIG.7A: Normal clotting system with the intrinsic and extrinsic cascadecomponents. FIG. 7B illustrates a variation in which an inactive/lowactive form of FVII-XTEN (FVII*) is intended to bypass the FIX and FVIIIcomponents of the intrinsic system when activated endogenously afteradministration.

FIG. 9 is a graph of the distribution of cell cluster size (gray bars)and FVII ELISA titers in ng/ml (black bars) by ELISA of clones fromprimary screening of pBC0014 CHO-K1 transformants (not all clones werelabeled underneath the bars due to insufficient space)(see Example 25for experimental details). Clones were sorted according to ELISA titerlow to high (left to right).

FIG. 10 is a graph of cell counts (white bars) and FVII titers in ng/ml(black bars) of the top pBC0014 clones (see Example 25 for experimentaldetails). Clones were sorted according to ELISA titer, low to high (leftto right).

FIG. 11 is a graph of the ratio of FVII titer over cell count of the toppBC0014 clones (see Example 25 for experimental details). Clones weresorted according to the ratio, low to high (left to right).

FIG. 12 is a Western blot of top pBC0014 clones according to ELISA,clotting, ELISA/cell count and clotting/cell count ratios (see Example25 for experimental details). Clone 6G1 expressed a truncated productand was not evaluated further.

FIG. 13 is a Western blot of the top pBC0016 clones according to ELISA,clotting, ELISA/cell count and clotting/cell count ratios (see Example25 for experimental details).

FIG. 14 is a Western blot of the top pBC0018 clones according to ELISA,clotting, ELISA/cell count and clotting/cell count ratios (see Example25 for experimental details). Clone 3B2 expressed a truncated productand was not evaluated further.

FIG. 15 shows purification of FVII-AE864 by anti-GLA affinitychromatography (see Example 26 for experimental details). SDS-PAGEanalysis demonstrating the purification of FVII-AE864 from concentratedsupernatant and the >90% purity of the EDTA eluted fractions.

FIG. 16 shows activation of FVII-XTEN fusions to FVIIa-XTEN fusions byFXa treatment (see Example 26 for experimental details). SDS-PAGEanalysis demonstrates the appearance of a light chain band underreducing conditions after FXa treatment, but not in the untreatedsample. Additionally, there is a downwards shift in the upper bandindicating the loss of the light chain.

FIG. 17 shows an SDS-PAGE demonstrating auto-activation of FVII-XTENfusions to FVIIa-XTEN fusions (see Example 26 for experimental details).SDS-PAGE analysis demonstrating appearance of a light chain band underreducing conditions after FXa treatment and after incubation at 4° C. athigh concentration with CaCl₂. Additionally, there is a downwards shiftin the upper band indicating the loss of the light chain.

FIG. 18 shows SEC Analysis of FVII-AE864 and FVII-AE288 (see Example 26for experimental details). The SEC shows a monodispersed population withminimal contamination and no aggregates at the void volume of the column(˜22 ml).

FIG. 19 shows the purification of FVII-AE864 by anion exchangechromatography (see Example 26 for experimental details). Thechromatograms depict the elution profiles of the total protein contentand the FVII activity from a Macrocap Q column with the bulk of theactivity eluting later than the contaminant proteins, creating a net5-fold purification.

FIG. 20 shows purification of FVII-AE864 by hydrophobic interactionchromatography (see Example 26 for experimental details). Thechromatograms depict the elution profiles of the total protein contentand the FVII activity from a toyopearl phenyl column with the bulk ofthe activity eluting earlier than the contaminant proteins, creating anet 2-fold purification.

FIG. 21 shows two chromatography outputs demonstrating removal ofaggregated protein from monomeric FVII-AE864 with anion exchangechromatography (see Example 26 for experimental details).

FIG. 21A is a chromatogram depicting the elution profile of FVII-XTENfrom a macrocap Q column with two peaks eluting after the buffer relatedearly peak. FIG. 21B shows SEC chromatograms of the early and latemacrocap Q peaks demonstrating the absence of aggregates in the earlypeak.

FIG. 22 shows results of ELISA or aPTT assays, showing FIX/cFXI/XTEN hasenhanced activity compared to FIX-XTEN (see Example 29 for experimentaldetails). Transiently expressed FIX constructs were assayed for antigencontent by ELISA and for activity by aPTT based assays. While theantigen content of FIX-XTEN was similar to the FIX/cFXI/XTEN constructsthe activity was significantly increased. This increase is attributed tothe specific action of the FXI protease in the assays as theFIX/cTEV/XTEN does not show a significantly different activity toFIX-XTEN. Note the ELISA titer of the FIX sample is 197 ng/ml and is offthe scale of the graph.

FIG. 23 shows the pharmacokinetic profile after a single doseadministered subcutaneously to rats, with the derived equivalent FVIIconcentration shown, as described in Example 30.

FIG. 24 shows the pharmacokinetic profile after a single doseadministered subcutaneously to rats, with the derived equivalent FIXconcentration shown, as described in Example 31.

FIG. 25 shows the pharmacokinetic profile (plasma concentrations) incynomolgus monkeys after single doses of different compositions of GFPlinked to unstructured polypeptides of varying length, administeredeither subcutaneously or intravenously, as described in Example 39. Thecompositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 andXTEN_AD836-GFP. Blood samples were analyzed at various times afterinjection and the concentration of GFP in plasma was measured by ELISAusing a polyclonal antibody against GFP for capture and a biotinylatedpreparation of the same polyclonal antibody for detection. Results arepresented as the plasma concentration versus time (h) after dosing andshow, in particular, a considerable increase in half-life for theXTEN_AD836-GFP, the composition with the longest sequence length ofXTEN. The construct with the shortest sequence length, the GFP-L288 hadthe shortest half-life.

FIG. 26 shows an SDS-PAGE gel of samples from a stability study of thefusion protein of XTEN_AE864 fused to the N-terminus of GFP (see Example40). The GFP-XTEN was incubated in cynomolgus plasma and rat kidneylysate for up to 7 days at 37° C. In addition, GFP-XTEN administered tocynomolgus monkeys was also assessed. Samples were withdrawn at 0, 1 and7 days and analyzed by SDS PAGE followed by detection using Westernanalysis with antibodies against GFP.

FIG. 27 shows three randomized libraries used for the third and fourthcodons in the N-terminal sequences of clones from LCW546, LCW547 andLCW552 (see Example 14 for experimental details). The libraries weredesigned with the third and fourth residues modified such that allcombinations of allowable XTEN codons were present at these positions,as shown. In order to include all the allowable XTEN codons for eachlibrary, nine pairs of oligonucleotides encoding 12 amino acids withcodon diversities of third and fourth residues were designed, annealedand ligated into the NdeI/BsaI restriction enzyme digested stuffervector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed into E. coliBL21Gold(DE3) competent cells to obtain colonies of the three librariesLCW0569 (SEQ ID NOS 773 and 774, respectively), LCW0570 (SEQ ID NOS 775and 776, respectively), and LCW0571 (SEQ ID NOS 777 and 778,respectively).

FIG. 28 shows a histogram of a retest of the top 75 clones after theoptimization step, as described in Example 15, for GFP fluorescencesignal, relative to the benchmark CBD_AM875 construct. The resultsindicated that several clones were now superior to the benchmark clones.

FIG. 29 is a schematic of a combinatorial approach undertaken for theunion of codon optimization preferences for two regions of theN-terminus 48 amino acids (see Example 16 for experimental details). Theapproach created novel 48mers at the N-terminus of the XTEN protein forevaluation of the optimization of expression that resulted in leadersequences that can be a solution for the expression of XTEN proteinswhere the XTEN is N-terminal to the CF.

FIG. 30 shows an SDS-PAGE gel confirming the expression of preferredclones obtained from the XTEN N-terminal codon optimization experiments,in comparison to benchmark XTEN clones comprising CBD leader sequencesat the N-terminus of the construct sequences, as described in Example17.

FIG. 31 is a schematic flowchart of representative steps in theassembly, production and the evaluation of a XTEN.

FIG. 32 is a schematic flowchart of representative steps in the assemblyof a CFXTEN polynucleotide construct encoding a fusion protein.Individual oligonucleotides 501 are annealed into sequence motifs 502such as a 12 amino acid motif (“12-mer”), which is subsequently ligatedwith an oligo containing BbsI, and KpnI restriction sites 503.Additional sequence motifs from a library are annealed to the 12-meruntil the desired length of the XTEN gene 504 is achieved. The XTEN geneis cloned into a stuffer vector. In this case, the vector encodes anoptional Flag sequence 506 followed by a stopper sequence that isflanked by BsaI, BbsI, and KpnI sites 507 and an FVII gene 508,resulting in the gene 500 encoding an XTEN-FVII fusion protein.

FIG. 33 is a schematic flowchart of representative steps in the assemblyof a gene encoding fusion protein comprising a CF and XTEN, itsexpression and recovery as a fusion protein, and its evaluation as acandidate CFXTEN product.

FIGS. 34A-34D are schematic representations of the design of CFXTENexpression vectors with different processing strategies. FIG. 34A showsan expression vector encoding XTEN fused to the 3′ end of the sequenceencoding FVII. Note that no additional leader sequences are required inthis vector. FIG. 34B depicts an expression vector encoding XTEN fusedto the 5′ end of the sequence encoding FVII with a CBD leader sequenceand a TEV protease site. FIG. 34C depicts an expression vector as inFIG. 34B where the CBD and TEV processing sites have been replaced withan optimized N-terminal leader sequence (NTS). FIG. 34D depicts anexpression vector encoding an NTS sequence, an XTEN, a sequence encodingVFII, and then a second sequence encoding an XTEN.

FIG. 35 shows results of a size exclusion chromatography analysis ofglucagon-XTEN construct samples measured against protein standards ofknown molecular weight, with the graph output as absorbance versusretention volume, as described in Example 37. The glucagon-XTENconstructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and4) glucagon-Y36. The results indicate an increase in apparent molecularweight with increasing length of XTEN moiety.

FIG. 36 shows sequence alignments between portions of native FIX, nativeFVII, and FVII-FIX sequence hybrids (SEQ ID NOS 779-786, respectively,in order of appearance) with different portions of the AP domainincorporated in the portion of the molecule spanning the EGF2 and Prodomains. The legend provides construct names. Gaps in an individualsequence (dashes) represents stretches of non-homology to FIX but areotherwise continuous, linked sequences. The underlined amino acids areFIX-derived sequence.

DETAILED DESCRIPTION OF THE INVENTION

Before the embodiments of the invention are described, it is to beunderstood that such embodiments are provided by way of example only,and that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.

Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including but not limited to boththe D or L optical isomers, and amino acid analogs and peptidomimetics.Standard single or three letter codes are used to designate amino acids.

The term “natural L-amino acid” means the L optical isomer forms ofglycine (G), proline (P), alanine (A), valine (V), leucine (L),isoleucine (I), methionine (M), cysteine (C), phenylalanine (F),tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R),glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D),serine (S), and threonine (T).

The term “non-naturally occurring,” as applied to sequences and as usedherein, means polypeptide or polynucleotide sequences that do not have acounterpart to, are not complementary to, or do not have a high degreeof homology with a wild-type or naturally-occurring sequence found in amammal. For example, a non-naturally occurring polypeptide or fragmentmay share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or evenless amino acid sequence identity as compared to a natural sequence whensuitably aligned.

The terms “hydrophilic” and “hydrophobic” refer to the degree ofaffinity that a substance has with water. A hydrophilic substance has astrong affinity for water, tending to dissolve in, mix with, or bewetted by water, while a hydrophobic substance substantially lacksaffinity for water, tending to repel and not absorb water and tendingnot to dissolve in or mix with or be wetted by water. Amino acids can becharacterized based on their hydrophobicity. A number of scales havebeen developed. An example is a scale developed by Levitt, M, et al., JMol Biol (1976) 104:59, which is listed in Hopp, T P, et al., Proc NatlAcad Sci USA (1981) 78:3824. Examples of “hydrophilic amino acids” arearginine, lysine, threonine, alanine, asparagine, and glutamine. Ofparticular interest are the hydrophilic amino acids aspartate,glutamate, and serine, and glycine. Examples of “hydrophobic aminoacids” are tryptophan, tyrosine, phenylalanine, methionine, leucine,isoleucine, and valine.

A “fragment” is a truncated form of a native biologically active proteinthat retains at least a portion of the therapeutic and/or biologicalactivity. A “variant” is a protein with sequence homology to the nativebiologically active protein that retains at least a portion of thetherapeutic and/or biological activity of the biologically activeprotein. For example, a variant protein may share at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identitycompared with the reference biologically active protein. As used herein,the term “biologically active protein moiety” includes proteins modifieddeliberately, as for example, by site directed mutagenesis, insertions,or accidentally through mutations.

As used herein, “internal XTEN” refers to XTEN sequences that have beeninserted into the sequence of the coagulation factor. Internal XTENs canbe constructed by insertion of an XTEN sequence into the sequence of acoagulation factor such as FIX or FVII, either by insertion between twoadjacent amino acids or domains of the coagulation factor or whereinXTEN replaces a partial, internal sequence of the coagulation factor.

As used herein, “terminal XTEN” refers to XTEN sequences that have beenfused to or in the N- or C-terminus of the coagulation factor or to aproteolytic cleavage sequence at the N- or C-terminus of the coagulationfactor. Terminal XTENs can be fused to the native termini of thecoagulation factor. Alternatively, terminal XTENs can replace a terminalsequence of the coagulation factor.

The term “XTEN release site” refers to a sequence in CFXTEN fusionproteins that can be recognized and cleaved by a mammalian protease,effecting release of an XTEN or a portion of an XTEN from the CFXTENfusion protein. As used herein, “mammalian protease” means a proteasethat normally exists in the body fluids, cells or tissues of a mammal.XTEN release sites can be engineered to be cleaved by various mammalianproteases (a.k.a. “XTEN release proteases”) such as FXIa, FXIIa,kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, MMP-12,MMP13, MMP-17, MMP-20, or any protease that is present during a clottingevent.

“Activity” as applied to form(s) of a CFXTEN polypeptide providedherein, refers to retention of a biological activity of the nativecoagulation factor, wherein “biological activity” refers to an in vitroor in vivo biological function or effect, including but not limited toeither receptor or ligand binding, enzymatic activity, or an effect oncoagulation generally known in the art for the coagulation factor.

A “therapeutic effect” as applied to form(s) of a CFXTEN polypeptideprovided herein, refers to a physiologic effect, including but notlimited to the curing, mitigation, reversal, amelioration or preventionof disease or conditions in humans or other animals, or to otherwiseenhance physical or mental wellbeing of humans or animals. A“therapeutically effective amount” means an amount of compound effectiveto prevent, alleviate, reverse or ameliorate symptoms of disease or acondition (e.g., a bleeding episode) or prolong the survival of thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient for the subject vectors. Host cells includeprogeny of a single host cell. The progeny may not necessarily becompletely identical (in morphology or in genomic of total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation. A host cell includes cells transfected in vivo witha vector of this invention.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. As is apparent to those of skill in the art,a non-naturally occurring polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, does not require “isolation” todistinguish it from its naturally occurring counterpart. In addition, a“concentrated”, “separated” or “diluted” polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, is distinguishablefrom its naturally occurring counterpart in that the concentration ornumber of molecules per volume is generally greater than that of itsnaturally occurring counterpart. In general, a polypeptide made byrecombinant means and expressed in a host cell is considered to be“isolated.”

An “isolated” polynucleotide or polypeptide-encoding nucleic acid orother polypeptide-encoding nucleic acid is a nucleic acid molecule thatis identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal or extra-chromosomal location differentfrom that of natural cells.

A “chimeric” protein contains at least one fusion polypeptide comprisingregions in a different position in the sequence than that which occursin nature. The regions may normally exist in separate proteins and arebrought together in the fusion polypeptide; or they may normally existin the same protein but are placed in a new arrangement in the fusionpolypeptide. A chimeric protein may be created, for example, by chemicalsynthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship.

“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeablyherein. These terms refer to the joining together of two or morechemical elements or components, by whatever means including chemicalconjugation or recombinant means. For example, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe sequence. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and in reading phase or in-frame. An“in-frame fusion” refers to the joining of two or more open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the correct reading frame of the original ORFs. Thus, theresulting recombinant fusion protein is a single protein containing twoor more segments that correspond to polypeptides encoded by the originalORFs (which segments are not normally so joined in nature).

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminus direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide. A“partial sequence” is a linear sequence of part of a polypeptide that isknown to comprise additional residues in one or both directions.

“Heterologous” means derived from a genotypically distinct entity fromthe rest of the entity to which it is being compared. For example, aglycine rich sequence removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous glycine rich sequence. The term “heterologous” asapplied to a polynucleotide, a polypeptide, means that thepolynucleotide or polypeptide is derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared.

The terms “polynucleotides”, “nucleic acids”, “nucleotides” and“oligonucleotides” are used interchangeably. They refer to a polymericform of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

The term “complement of a polynucleotide” denotes a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence, such that it could hybridize with areference sequence with complete fidelity.

“Recombinant” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of in vitrocloning, restriction and/or ligation steps, and other procedures thatresult in a construct that can potentially be expressed in a host cell.

The terms “gene” and “gene fragment” are used interchangeably herein.They refer to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated. A gene or gene fragment may be genomic orcDNA, as long as the polynucleotide contains at least one open readingframe, which may cover the entire coding region or a segment thereof. A“fusion gene” is a gene composed of at least two heterologouspolynucleotides that are linked together.

“Homology” or “homologous” refers to sequence similarity orinterchangeability between two or more polynucleotide sequences or twoor more polypeptide sequences. When using a program such as BestFit todetermine sequence identity, similarity or homology between twodifferent amino acid sequences, the default settings may be used, or anappropriate scoring matrix, such as blosum45 or blosum80, may beselected to optimize identity, similarity or homology scores.Preferably, polynucleotides that are homologous are those whichhybridize under stringent conditions as defined herein and have at least70%, preferably at least 80%, more preferably at least 90%, morepreferably 95%, more preferably 97%, more preferably 98%, and even morepreferably 99% sequence identity compared to those sequences.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments or genes, linking them together. To ligatethe DNA fragments or genes together, the ends of the DNA must becompatible with each other. In some cases, the ends will be directlycompatible after endonuclease digestion. However, it may be necessary tofirst convert the staggered ends commonly produced after endonucleasedigestion to blunt ends to make them compatible for ligation.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Generally,stringency of hybridization is expressed, in part, with reference to thetemperature and salt concentration under which the wash step is carriedout. Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short polynucleotides (e.g., 10to 50 nucleotides) and at least about 60° C. for long polynucleotides(e.g., greater than 50 nucleotides)—for example, “stringent conditions”can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C.,and three washes for 15 min each in 0.1×SSC/1% SDS at 60° C. to 65° C.Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C.may be used. SSC concentration may be varied from about 0.1 to 2×SSC,with SDS being present at about 0.1%. Such wash temperatures aretypically selected to be about 5° C. to 20° C. lower than the thermalmelting point for the specific sequence at a defined ionic strength andpH. The Tm is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. An equation for calculating Tm and conditions for nucleic acidhybridization are well known and can be found in Sambrook, J. et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Press, Plainview N.Y.; specifically see volume 2 andchapter 9. Typically, blocking reagents are used to block non-specifichybridization. Such blocking reagents include, for instance, sheared anddenatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, suchas formamide at a concentration of about 35-50% v/v, may also be usedunder particular circumstances, such as for RNA:DNA hybridizations.Useful variations on these wash conditions will be readily apparent tothose of ordinary skill in the art.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Percent identity may bemeasured over the length of an entire defined polynucleotide sequence,or may be measured over a shorter length, for example, over the lengthof a fragment taken from a larger, defined polynucleotide sequence, forinstance, a fragment of at least 45, at least 60, at least 90, at least120, at least 150, at least 210 or at least 450 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Percent (%) sequence identity,” with respect to the polypeptidesequences identified herein, is defined as the percentage of amino acidresidues in a query sequence that are identical with the amino acidresidues of a second, reference polypeptide sequence or a portionthereof, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Percentidentity may be measured over the length of an entire definedpolypeptide sequence, or may be measured over a shorter length, forexample, over the length of a fragment taken from a larger, definedpolypeptide sequence, for instance, a fragment of at least 15, at least20, at least 30, at least 40, at least 50, at least 70 or at least 150contiguous residues. Such lengths are exemplary only, and it isunderstood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listing, may be used todescribe a length over which percentage identity may be measured.

The term “non-repetitiveness” as used herein in the context of apolypeptide refers to a lack or limited degree of internal homology in apeptide or polypeptide sequence. The term “substantially non-repetitive”can mean, for example, that there are few or no instances of fourcontiguous amino acids in the sequence that are identical amino acidtypes or that the polypeptide has a subsequence score (defined infra) of10 or less or that there isn't a pattern in the order, from N- toC-terminus, of the sequence motifs that constitute the polypeptidesequence. The term “repetitiveness” as used herein in the context of apolypeptide refers to the degree of internal homology in a peptide orpolypeptide sequence. In contrast, a “repetitive” sequence may containmultiple identical copies of short amino acid sequences. For instance, apolypeptide sequence of interest may be divided into n-mer sequences andthe number of identical sequences can be counted. Highly repetitivesequences contain a large fraction of identical sequences whilenon-repetitive sequences contain few identical sequences. In the contextof a polypeptide, a sequence can contain multiple copies of shortersequences of defined or variable length, or motifs, in which the motifsthemselves have non-repetitive sequences, rendering the full-lengthpolypeptide substantially non-repetitive. The length of polypeptidewithin which the non-repetitiveness is measured can vary from 3 aminoacids to about 200 amino acids, about from 6 to about 50 amino acids, orfrom about 9 to about 14 amino acids. “Repetitiveness” used in thecontext of polynucleotide sequences refers to the degree of internalhomology in the sequence such as, for example, the frequency ofidentical nucleotide sequences of a given length. Repetitiveness can,for example, be measured by analyzing the frequency of identicalsequences.

A “vector” is a nucleic acid molecule, preferably self-replicating in anappropriate host, which transfers an inserted nucleic acid molecule intoand/or between host cells. The term includes vectors that functionprimarily for insertion of DNA or RNA into a cell, replication ofvectors that function primarily for the replication of DNA or RNA, andexpression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions. An “expression vector” is a polynucleotide which,when introduced into an appropriate host cell, can be transcribed andtranslated into a polypeptide(s). An “expression system” usuallyconnotes a suitable host cell comprised of an expression vector that canfunction to yield a desired expression product.

“Serum degradation resistance,” as applied to a polypeptide, refers tothe ability of the polypeptides to withstand degradation in blood orcomponents thereof, which typically involves proteases in the serum orplasma. The serum degradation resistance can be measured by combiningthe protein with human (or mouse, rat, monkey, as appropriate) serum orplasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16days), typically at about 37° C. The samples for these time points canbe run on a Western blot assay and the protein is detected with anantibody. The antibody can be to a tag in the protein. If the proteinshows a single band on the western, where the protein's size isidentical to that of the injected protein, then no degradation hasoccurred. In this exemplary method, the time point where 50% of theprotein is degraded, as judged by Western blots or equivalenttechniques, is the serum degradation half-life or “serum half-life” ofthe protein.

The term “t_(1/2)” as used herein means the terminal half-lifecalculated as ln(2)/K_(el). K_(el) is the terminal elimination rateconstant calculated by linear regression of the terminal linear portionof the log concentration vs. time curve. Half-life typically refers tothe time required for half the quantity of an administered substancedeposited in a living organism to be metabolized or eliminated by normalbiological processes. The terms “t_(1/2)”, “terminal half-life”,“elimination half-life” and “circulating half-life” are usedinterchangeably herein.

“Active clearance” means the mechanisms by which CF is removed from thecirculation other than by filtration or coagulation, and which includesremoval from the circulation mediated by cells, receptors, metabolism,or degradation of the CF.

“Apparent molecular weight factor” and “apparent molecular weight” arerelated terms referring to a measure of the relative increase ordecrease in apparent molecular weight exhibited by a particular aminoacid sequence. The apparent molecular weight is determined using sizeexclusion chromatography (SEC) and similar methods compared to globularprotein standards and is measured in “apparent kD” units. The apparentmolecular weight factor is the ratio between the apparent molecularweight and the actual molecular weight; the latter predicted by adding,based on amino acid composition, the calculated molecular weight of eachtype of amino acid in the composition or by estimation from comparisonto molecular weight standards in an SDS electrophoresis gel.

The terms “hydrodynamic radius” or “Stokes radius” is the effectiveradius (Rh in nm) of a molecule in a solution measured by assuming thatit is a body moving through the solution and resisted by the solution'sviscosity. In the embodiments of the invention, the hydrodynamic radiusmeasurements of the XTEN fusion proteins correlate with the ‘apparentmolecular weight factor’, which is a more intuitive measure. The“hydrodynamic radius” of a protein affects its rate of diffusion inaqueous solution as well as its ability to migrate in gels ofmacromolecules. The hydrodynamic radius of a protein is determined byits molecular weight as well as by its structure, including shape andcompactness. Methods for determining the hydrodynamic radius are wellknown in the art, such as by the use of size exclusion chromatography(SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Mostproteins have globular structure, which is the most compactthree-dimensional structure a protein can have with the smallesthydrodynamic radius. Some proteins adopt a random and open,unstructured, or ‘linear’ conformation and as a result have a muchlarger hydrodynamic radius compared to typical globular proteins ofsimilar molecular weight.

“Physiological conditions” refers to a set of conditions in a livinghost as well as in vitro conditions, including temperature, saltconcentration, pH, that mimic those conditions of a living subject. Ahost of physiologically relevant conditions for use in in vitro assayshave been established. Generally, a physiological buffer contains aphysiological concentration of salt and is adjusted to a neutral pHranging from about 6.5 to about 7.8, and preferably from about 7.0 toabout 7.5. A variety of physiological buffers are listed in Sambrook etal. (1989). Physiologically relevant temperature ranges from about 25°C. to about 38° C., and preferably from about 35° C. to about 37° C.

A “reactive group” is a chemical structure that can be coupled to asecond reactive group. Examples for reactive groups are amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups,azide groups. Some reactive groups can be activated to facilitatecoupling with a second reactive group. Non-limiting examples foractivation are the reaction of a carboxyl group with carbodiimide, theconversion of a carboxyl group into an activated ester, or theconversion of a carboxyl group into an azide function.

“Controlled release agent”, “slow release agent”, “depot formulation”and “sustained release agent” are used interchangeably to refer to anagent capable of extending the duration of release of a polypeptide ofthe invention relative to the duration of release when the polypeptideis administered in the absence of agent. Different embodiments of thepresent invention may have different release rates, resulting indifferent therapeutic amounts.

The terms “antigen”, “target antigen” and “immunogen” are usedinterchangeably herein to refer to the structure or binding determinantthat an antibody fragment or an antibody fragment-based therapeuticbinds to or has specificity against.

The term “payload” as used herein refers to a protein or peptidesequence that has biological or therapeutic activity; the counterpart tothe pharmacophore of small molecules. Examples of payloads include, butare not limited to, cytokines, enzymes, hormones and blood and growthfactors. Payloads can further comprise genetically fused or chemicallyconjugated moieties such as chemotherapeutic agents, antiviralcompounds, toxins, or contrast agents. These conjugated moieties can bejoined to the rest of the polypeptide via a linker that may be cleavableor non-cleavable.

The term “antagonist”, as used herein, includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of a native polypeptide disclosed herein. Methods foridentifying antagonists of a polypeptide may comprise contacting anative polypeptide with a candidate antagonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the native polypeptide. In the context of the presentinvention, antagonists may include proteins, nucleic acids,carbohydrates, antibodies or any other molecules that decrease theeffect of a biologically active protein.

The term “agonist” is used in the broadest sense and includes anymolecule that mimics a biological activity of a native polypeptidedisclosed herein. Suitable agonist molecules specifically includeagonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides, peptides, small organicmolecules, etc. Methods for identifying agonists of a native polypeptidemay comprise contacting a native polypeptide with a candidate agonistmolecule and measuring a detectable change in one or more biologicalactivities normally associated with the native polypeptide.

“Activity” for the purposes herein refers to an action or effect of acomponent of a fusion protein consistent with that of the correspondingnative biologically active protein, wherein “biological activity” refersto an in vitro or in vivo biological function or effect, including butnot limited to receptor binding, antagonist activity, agonist activity,or a cellular or physiologic response.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” is used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a subjectat risk of developing a particular disease, or to a subject reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect”, as used herein, refers to a physiologic effect,including but not limited to the cure, mitigation, amelioration, orprevention of disease in humans or other animals, or to otherwiseenhance physical or mental wellbeing of humans or animals, caused by afusion polypeptide of the invention other than the ability to induce theproduction of an antibody against an antigenic epitope possessed by thebiologically active protein. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose”, as used herein, refer to an amount of a biologicallyactive protein, either alone or as a part of a fusion proteincomposition, that is capable of having any detectable, beneficial effecton any symptom, aspect, measured parameter or characteristics of adisease state or condition when administered in one or repeated doses toa subject. Such effect need not be absolute to be beneficial.

The term “therapeutically effective dose regimen”, as used herein,refers to a schedule for consecutively administered multiple doses(i.e., at least two or more) of a biologically active protein, eitheralone or as a part of a fusion protein composition, wherein the dosesare given in therapeutically effective amounts to result in sustainedbeneficial effect on any symptom, aspect, measured parameter orcharacteristics of a disease state or condition.

I). General Techniques

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook, J.et al., “Molecular Cloning: A Laboratory Manual,” 3^(rd) edition, ColdSpring Harbor Laboratory Press, 2001; “Current protocols in molecularbiology”, F. M. Ausubel, et al. eds., 1987; the series “Methods inEnzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practicalapproach”, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., OxfordUniversity Press, 1995; “Antibodies, a laboratory manual” Harlow, E. andLane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics,” 11^(th) Edition,McGraw-Hill, 2005; and Freshney, R. I., “Culture of Animal Cells: AManual of Basic Technique,”4^(th) edition, John Wiley & Sons, Somerset,N J, 2000, the contents of which are incorporated in their entiretyherein by reference.

II). Coagulation Factors

The present invention relates in part to fusion protein compositionscomprising coagulation factors (CF). As used herein, “coagulationfactor” or “CF” refers to factor IX (FIX), factor VII (FVII), sequencecombinations of FVII and FIX, or mimetics, sequence variants andtruncated versions thereof.

(a) Factor IX

“Factor IX” or “FIX” means a coagulation factor protein and species andsequence variants thereof, and includes, but is not limited to, the 461single-chain amino acid sequence of human FIX precursor polypeptide(“prepro”) and the 415 single-chain amino acid sequence of mature humanFIX. FIX includes any form of factor IX molecule with the typicalcharacteristics of blood coagulation factor IX. As used herein “factorIX” and “FIX” are intended to encompass polypeptides that comprise thedomains Gla (region containing γ-carboxyglutamic acid residues), EGF1and EGF2 (region containing sequences homologous to human epidermalgrowth factor), activation peptide (formed by residues R136-R180 of themature FIX), and the C-terminal protease domain (“Pro”), or synonyms ofthese domains known in the art, or can be a truncated fragment or asequence variant that retains at least a portion of the biologicalactivity of the native protein. FIX or sequence variants have beencloned, as described in U.S. Pat. Nos. 4,770,999, 7,700,734, and cDNAcoding for human factor IX has been isolated, characterized, and clonedinto expression vectors (see, for example, Choo et al., Nature299:178-180 (1982); Fair et al., Blood 64:194-204 (1984); and Kurachi etal., Proc. Natl. Acad. Sci., U.S.A. 79:6461-6464 (1982)).

Human factor IX (FIX) is encoded by a single-copy gene residing on theX-chromosome at q27.1. The human FIX mRNA is composed of 205 bases forthe 5′ untranslated region, 1383 bases for the prepro factor IX, a stopcodon and 1392 bases for the 3′ untranslated region. The FIX polypeptideis 55 kDa, synthesized as a prepropolypetide chain composed of threeregions: a signal peptide of 28 amino acids, a propeptide of 18 aminoacids, which is required for gamma-carboxylation of glutamic acidresidues, and a mature factor IX of 415 amino acids. The propeptide isan 18-amino acid residue sequence N-terminal to thegamma-carboxyglutamate domain. The propeptide binds vitamin K-dependentgamma carboxylase and then is cleaved from the precursor polypeptide ofFIX by an endogenous protease, most likely PACE (paired basic amino acidcleaving enzyme), also known as furin or PCSK3. Without the gammacarboxylation, the Gla domain is unable to bind calcium to assume thecorrect conformation necessary to anchor the protein to negativelycharged phospholipid surfaces, thereby rendering factor IXnonfunctional. Even if it is carboxylated, the Gla domain also dependson cleavage of the propeptide for proper function, since retainedpropeptide interferes with conformational changes of the Gla domainnecessary for optimal binding to calcium and phospholipid. In humans,the resulting mature factor IX is secreted by liver cells into the bloodstream as an inactive zymogen, a single chain protein of 415 amino acidresidues that contains approximately 17% carbohydrate by weight(Schmidt, A. E., et al. (2003) Trends Cardiovasc Med, 13: 39). Themature factor IX is composed of several domains that in an N- toC-terminus configuration are: a Gla domain, an EGF1 domain, an EGF2domain, an activation peptide (AP) domain, and a protease (or catalytic)domain. FIX contains two activation peptides formed by R145-A146 andR180-V181, respectively. Following activation, the single-chain FIXbecomes a 2-chain molecule, in which the two chains are linked by adisulfide bond attaching the enzyme to the Gla domain. CFs can beengineered by replacing their activation peptides resulting in alteredactivation specificity. In mammals, mature FIX must be activated byactivated factor XI to yield factor IXa. The protease domain provides,upon activation of FIX to FIXa, the catalytic activity of FIX. Activatedfactor VIII (FVIIIa) is the specific cofactor for the full expression ofFIXa activity.

Proteins involved in clotting include factor I, factor II, factor III,factor IV, factor V, factor VI, factor VII, factor VIII, factor IX,factor X, factor XI, factor XII, factor XIII, Protein C, and tissuefactor (“clotting proteins”). The majority of the clotting proteins ispresent in zymogen form that when activated exhibits a pro-coagulantprotease activity to activate other clotting proteins, contributing tothe intrinsic or extrinsic coagulation pathway and clot formation. Inthe intrinsic pathway of the coagulation cascade, FIX associates with acomplex of activated factor VIII, factor X, calcium, and phospholipid.In the complex, FIX is activated by factor XIa. The activation of factorIX is achieved by a two-step removal of the activation peptide (Ala146-Arg 180) from the molecule (Bajaj et al., Human factor IX and factorIXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is made at theArg 145-Ala 146 site by either factor XIa or factor VIIa/tissue factor.The second and rate limiting cleavage is made at Arg 180-Val 181. Theactivation removes 35 residues. Activated human factor IX exists as aheterodimer of the C-terminal heavy chain (28 kDa) and an N-terminallight chain (18 kDa), which are held together by one disulfide bridgeattaching the enzyme to the Gla domain. Factor IXa in turn activatesfactor X in concert with activated factor VIII. Alternatively, factorsIX and X can both be activated by factor VIIa complexed with lipidatedTissue Factor, generated via the extrinsic pathway. Factor Xa thenparticipates in the final common pathway whereby prothrombin isconverted to thrombin, and thrombin in turn converts fibrinogen tofibrin to form the clot.

Defects in the coagulation process can lead to bleeding disorders inwhich the time taken for clot formation is prolonged. Such defects canbe congenital or acquired. For example, hemophilia A and B are inheriteddiseases characterized by deficiencies in factor VIII (FVIII) and FIX,respectively. Replacement therapy with these proteins, generallyprepared as recombinant proteins, may be used in the therapeuticintervention of hemophilia B (Christmas Disease) and factor IX-relatedbleeding disorders. Factor IX can be used in the treatment of bothconditions. In some cases, however, patients develop antibodies againstthe administered proteins that reduce or negate the efficacy of thetreatment.

The invention contemplates inclusion of FIX sequences in the CFXTENcompositions that have homology to FIX sequences, sequence fragmentsthat are natural, such as from humans, non-human primates, mammals(including domestic animals), and non-natural sequence variants whichretain at least a portion of the biologic activity or biologicalfunction of FIX and/or that are useful for preventing, treating,mediating, or ameliorating a coagulation factor-related disease,deficiency, disorder or condition (e.g., bleeding episodes related totrauma, surgery, of deficiency of a coagulation factor). Sequences withhomology to human FIX can be found by standard homology searchingtechniques, such as NCBI BLAST.

In one embodiment, the FIX incorporated into the subject compositions isa recombinant polypeptide with a sequence corresponding to a proteinfound in nature. In another embodiment, the FIX is a sequence variant,fragment, homolog, or a mimetics of a natural sequence that retains atleast a portion of the biological activity of the corresponding nativeFIX. Table 1 provides a non-limiting list of amino acid sequences of FIXthat are encompassed by the CFXTEN fusion proteins of the invention. Anyof the FIX sequences or homologous derivatives to be incorporated intothe fusion protein compositions can be constructed by shufflingindividual mutations between the amino acid sequences of Table 1 andevaluated for activity. Those that retain at least a portion of thebiological activity of the native FIX are useful for the fusion proteincompositions of this invention. FIX that can be incorporated into aCFXTEN fusion protein includes a protein that has at least about 80%sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity compared to an amino acid sequence selected from Table1.

TABLE 1 FIX amino acid and nucleic acid sequences SEQ ID Name NO:Amino Acid Sequence FIX precursor 7MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF polypeptideVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT FIX Homo 8YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN sapiensPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 4 9MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF from PatentVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK USDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL 20080214462AENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 6 10MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF from PatentVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK USDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL 20080214462AENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 8 11MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF from PatentVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK USDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEG 20080214462YRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDATCLRSTKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEGCAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 2 12MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFV from PatentQGNLERECMEEKCSFEEPREVFENIEKIIEFWKQYVDGDQCESNPCLNGGSCKD US 7125841DINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNPTEAETILDNITQGTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRAIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWARVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 1 13YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 2 14YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 3 15YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 4 16YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 5 17YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 6 18YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 8 19YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN from PatentPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV USCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI 20080167219LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEEIEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

(b) Factor VII.

“Factor VII” or “FVII” means a coagulation factor protein and speciesand sequence variants thereof, and includes, but is not limited to, boththe inactive and activated forms (unless indicated to the contrary) ofthe 406 single-chain amino acid sequence of human FVII, and the 444amino acid sequence of the precursor protein. As used herein, factor VIIand FVII encompass polypeptides that comprise the domains Gla (regioncontaining γ-carboxyglutamic acid residues), EGF1 and EGF2 (regioncontaining sequences homologous to human epidermal growth factor), anactivation peptide domain that spans the sequence between the EGF2 andPro domains, and a catalytic or peptidase S1 domain (“Pro” regioncontaining the serine protease catalytic triad), or synonyms of thesedomains known in the art, or can be a truncated fragment or a sequencevariant that retains at least a portion of the biological activity ofthe native protein. Factor VII (FVII), a vitamin K-dependent plasmaprotein produced by the liver, initially circulates in the blood as azymogen. The main role of factor VII is to initiate the process ofcoagulation in conjunction with tissue factor (TF). Upon vessel injury,tissue factor is exposed to the blood and circulating factor VII. Oncebound to TF, FVII is activated to become the activated form of factorVII (FVIIa) by different proteases, among which are thrombin (factorIIa), factor Xa, IXa, XIIa, and the FVIIa-TF complex itself. The FVIIzymogen is activated by proteolytic cleavage at a single site,Arg¹⁵²-Ile¹⁵³, resulting in a two-chain protease linked by a singledisulphide bond (FVIIa). FVIIa binds its cofactor, tissue factor (TF),to form a complex which can activate factor X (FX) to FXa, therebyinitiating a coagulation cascade that results in fibrin formation andhemostasis. The complete nucleotide and amino acid sequences for humanfactor VII are known, and human FVII or sequence variants have beencloned, as described in U.S. Pat. Nos. 4,784,950, 5,833,982, 6,911,323,and 7,026,524.

Current therapeutic uses of factor VII exist but can be problematic inthe treatment of individuals exhibiting a deficiency in factor VII,factor VIII, or factor IX, and individuals with Von Willebrand's diseasewith FVIIa formulations. More specifically, individuals receivingfactors VIII and IX in replacement therapy frequently develop antibodiesto these proteins. Continuing treatment is exceedingly difficult becauseof the presence of these antibodies. Patients experiencing this problemare normally treated with an activated prothrombin complex known toconsist of a mixture of active and inactive clotting enzymes, includingfactor VIIa. FVII also is utilized in connection with treatment ofuncontrolled bleedings, such as trauma, and it is believed that factorVIIa is capable of activating factor X to factor Xa without binding totissue factor, and this activation reaction is believed to occurprimarily on activated blood platelets (Hedner et al. Blood Coagulation& Fibrinolysis, 2000; 11; 107-111).

Sequence variants of factor VII, whether exhibiting substantially thesame or better bioactivity than wild-type factor VII, or, alternatively,exhibiting substantially modified or reduced bioactivity relative towild-type factor VII, include, polypeptides having an amino acidsequence that differs from the sequence of wild-type factor VII byinsertion, deletion, or substitution of one or more amino acids. SuchFVII variants are known in the art, including those described in U.S.Pat. Nos. 6,960,657, 7,176,288, 7,414,022, 7,700,733, 20060205036A1,20080318276A1, and 20090011992A1, which are incorporated herein byreference.

Recombinant FVIIa has been approved for the treatment of hemophilia A orB patients that have inhibitors to FVIII or FIX, and also is used tostop bleeding episodes or prevent bleeding associated with trauma and/orsurgery. Recombinant FVIIa also has been approved for the treatment ofpatients with congenital FVII deficiency, and is increasingly beingutilized in off-label uses, such as the treatment of bleeding associatedwith other congenital or acquired bleeding disorders, trauma, andsurgery in hemophilic and non-hemophilic patients.

The invention contemplates inclusion in the CFXTEN compositionssequences with homology to FVII sequences, sequence fragments, mimeticsand non-natural sequence variants which retain at least a portion of thebiologic activity or biological function of FVIIa that are useful forpreventing, treating, mediating, or ameliorating a CF-related disease,deficiency, disorder or condition. In addition, because of thecomparatively long-half life of CFXTEN comprising FVII, compositionscomprising the inactive form of FVII that can be activated by mammalianendogenous proteases (described more fully below) or undergoautoactivation represents a means to treat subjects with certain formsof chronic coagulopathies with what is essentially a “prodrug” form ofFVII. Table 2 provides a list of sequences of FVII that are encompassedby the CFXTEN fusion proteins of the invention. FVII sequences orhomologous derivatives constructed by shuffling individual mutationsbetween species or families that retain at least a portion of thebiological activity of the native CF are useful for the fusion proteinsof this invention. FVII that can be incorporated into a CFXTEN fusionprotein include a protein that exhibits at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity compared to a sequence selected from Table 2.

TABLE 2 Factor VII amino acid sequences SEQ ID Name NO:Amino Acid Sequence FVII 20MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERE precursorCKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICF polypeptideCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRS EPRPGVLLRAPFPHuman FVII 21 ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQ(mature) NGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 22NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKSLTRNGPLKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 23NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKSMTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 24NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKCGQRLRKSKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 25NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIKPRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 26NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKKLTRAETVKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 27NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 28NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIQIRSVAKKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 29NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant 30ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIEPRSPSQKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP

III). Coagulation Factor Fusion Protein Compositions

The present invention provides fusion protein compositions comprisingcoagulation factors (CF). One way to increase the circulation half-lifeof a therapeutic protein is to reduce the renal clearance of theprotein. This may be achieved by conjugating the protein to a polymerthat s capable of conferring an increased molecular size (orhydrodynamic radius) to the protein, and hence, reduces renal clearance.Thus, one object of the present invention is to provide improved FIX orFVII (or FVIIa) molecules with a longer circulation, or terminalhalf-life (thereby decreasing the number of necessary administrations)and that retain at least a portion of the activity of the nativecoagulation factors, thereby to treat coagulation deficiencies anduncontrolled bleedings more efficiently. In one aspect, the inventionprovides isolated monomeric fusion proteins of CF comprising thefull-length sequence or sequence variants of a CF, such as FIX or FVII,covalently linked to extended recombinant polypeptides (“XTEN” or“XTENs”). As described more fully below, the fusion proteins optionallyinclude spacer sequences that further comprise cleavage sequences torelease the CF from the fusion protein when acted on by a protease.

In one aspect, the invention provides an isolated fusion proteincomprising at least a first biologically active coagulation factorprotein covalently linked to one or more extended recombinantpolypeptides (“XTEN”), resulting in a fusion protein composition(hereinafter “CFXTEN”). The term “CFXTEN”, as used herein, is meant toencompass fusion polypeptides that comprise one or more payload regionseach comprising a biologically active CF that mediates one or morebiological or therapeutic activities associated with a coagulationfactor and at least one other region comprising at least a first XTENpolypeptide that serves as a carrier. In one embodiment, the coagulationfactor is FIX or a sequence variant of FIX, as disclosed above(including sequences with homology to the sequences of Table 1). Inanother embodiment, the coagulation factor is FVII, which can includethe activated form of FVII, or a sequence variant of FVII, as disclosedabove (including sequences with homology with the sequences of Table 2).In the case of CFXTEN compositions of the invention comprising FVII,activation of the FVII component may be carried out by exposure toactivated factor X, by auto-activation, or according to procedures knownin the art, such as those disclosed by Osterud, et al., Biochemistry11:2853-2857 (1972); Thomas, U.S. Pat. No. 4,456,591; Hedner and Kisiel,J. Clin. Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, BehringInst. Mitt. 73:29-42 (1983). Alternatively, factor VII can be activatedby passing it through an ion-exchange chromatography column (see, e.g.,Bjoern et al. Research Disclosure (1986) 269:564-565), such as Mono Q(Pharmacia fine Chemicals) or similar chromatography resins.

The CF of the subject compositions, particularly those disclosed inTables 1 and 2, together with their corresponding nucleic acid and aminoacid sequences, are well known in the art and descriptions and sequencesare available in public databases such as Chemical Abstracts ServicesDatabases (e.g., the CAS Registry), GenBank, The Universal ProteinResource (UniProt) and subscription provided databases such as GenSeq(e.g., Derwent). Polynucleotide sequences may be a wild typepolynucleotide sequence encoding a given CF (e.g., either full length ormature), or in some instances the sequence may be a variant of the wildtype polynucleotide sequence (e.g., a polynucleotide which encodes thewild type biologically active protein, wherein the DNA sequence of thepolynucleotide has been optimized, for example, for expression in aparticular species; or a polynucleotide encoding a variant of the wildtype protein, such as a site directed mutant or an allelic variant. Itis well within the ability of the skilled artisan to use a wild-type orconsensus cDNA sequence or a codon-optimized variant of a CF to createCFXTEN constructs contemplated by the invention using methods known inthe art and/or in conjunction with the guidance and methods providedherein, and described more fully in the Examples.

The CF for inclusion in the CFXTEN of the invention include coagulationfactors or sequence variants that are useful, when administered to asubject, for mediating or preventing or ameliorating a disease, disorderor condition associated with bleeding disorders, coagulation factordeficiencies or defects in a coagulation factor. Of particular interestare CFXTEN fusion protein compositions for which an increase in apharmacokinetic parameter, increased solubility, increased stability, orsome other enhanced pharmaceutical property compared to native CF issought, or for which increasing the terminal half-life would improveefficacy, safety, or result in reduced dosing frequency and/or improvepatient compliance. Thus, the CFXTEN fusion protein compositions areprepared with various objectives in mind, including improving thetherapeutic efficacy of the bioactive CF by, for example, increasing thein vivo exposure or the length that the CFXTEN remains within thetherapeutic window when administered to a subject, compared to a CF notlinked to XTEN.

In one embodiment, the CF incorporated into the subject compositions canbe a recombinant polypeptide with a sequence corresponding to a proteinfound in nature. In another embodiment, the CF is a sequence variant,fragment, homolog, or mimetic of a natural sequence that retain at leasta portion of the biological activity of the native CF. In non-limitingexamples, a CF is a sequence that exhibits at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or100% sequence identity compared to a protein sequence selected fromTable 1 or from Table 2. In one embodiment, a CFXTEN fusion proteincomprises a single CF molecule linked to a single XTEN (e.g., an XTEN asdescribed more fully below). In another embodiment, the CFXTEN comprisesa first CF and a second molecule of the same CF, resulting in a fusionprotein comprising the two CF linked to one or more XTEN in an N- toC-terminus configuration selected from Table 6. In another embodiment,the CFXTEN fusion protein comprises a single CF molecule linked to afirst and a second XTEN, in which the CF is a sequence that exhibits atleast about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least about 99%, or 100% sequence identity compared to a proteinsequence selected from Table 1 or from Table 2, and the first and/or thesecond XTEN are sequences that exhibits at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or100% sequence identity compared to a sequence selected from Table 4.

The subject CFXTEN of the present invention exhibits an enhancement ofone or more pharmacokinetic parameters compared to the native CF. TheCFXTEN with enhanced pharmacokinetic parameters permits less frequentdosing or an enhanced pharmacologic effect, including but not limited tomaintaining the biologically active CFXTEN within the therapeutic windowbetween the minimum effective dose or blood concentration (C_(min)) andthe maximum tolerated dose or blood concentration (C_(max)) for a longerperiod of time compared to the CF not linked to XTEN. In such cases, thelinking of the CF to a fusion protein comprising a select XTENsequence(s) can result in an improvement in these properties, makingthem more useful as therapeutic or preventive agents compared to CF notlinked to XTEN. In some embodiments, the subject CFXTEN of the presentinvention has a cleavage sequence incorporated between the CF and theXTEN and the biologic activity of the CF component is enhanced by therelease of the CF from the fusion protein by cleavage of the cleavagesequence by an endogenous protease, as described below.

IV). Xtended Recombinant Polypeptides

In one aspect, the invention provides XTEN polypeptide compositions thatare useful as a fusion protein partner to which CF is linked, resultingin a CFXTEN fusion protein. XTEN are generally extended lengthpolypeptides with non-naturally occurring, substantially non-repetitivesequences that are composed mainly of small hydrophilic amino acids,with the sequence having a low degree or no secondary or tertiarystructure under physiologic conditions.

XTENs have utility as a fusion protein partners in that they serve as a“carrier,” conferring certain desirable pharmacokinetic, physicochemicaland pharmaceutical properties when linked to a CF protein to a create afusion protein. Such desirable properties include but are not limited toenhanced pharmacokinetic parameters and solubility characteristics ofthe compositions, amongst other properties described herein. Such fusionprotein compositions have utility to treat certain coagulationfactor-related diseases, disorders or conditions, as described herein.As used herein, “XTEN” specifically excludes whole antibodies orantibody fragments (e.g. single-chain antibodies and Fc fragments).

In some embodiments, the XTEN is a long polypeptide having greater thanabout 100 to about 3000 amino acid residues when used as a carrier orgreater than 400 to about 3000 residues cumulatively when more than oneXTEN unit is used in a single fusion protein. In other embodiments, whenXTEN is used as a linker between fusion protein components or where anincrease in half-life of the fusion protein is not needed but where anincrease in solubility or some other physico/chemical property for theCF fusion partner component is desired, an XTEN sequence shorter than100 amino acid residues, such as about 96, or about 84, or about 72, orabout 60, or about 48, or about 36 amino acid residues are incorporatedinto a fusion protein composition with the CF to effect the property.

The selection criteria for the XTEN to be linked to the biologicallyactive proteins used to create the inventive fusion proteinscompositions generally relate to attributes of physical/chemicalproperties and conformational structure of the XTEN that is, in turn,used to confer enhanced pharmaceutical and pharmacokinetic properties tothe fusion proteins compositions. The XTEN of the present inventionexhibits one or more of the following advantageous properties:conformational flexibility, enhanced aqueous solubility, high degree ofprotease resistance, low immunogenicity, low binding to mammalianreceptors, and increased hydrodynamic (or Stokes) radii; properties thatmake them particularly useful as fusion protein partners. Non-limitingexamples of the properties of the fusion proteins comprising CF that areenhanced by XTEN include increases in the overall solubility and/ormetabolic stability, reduced susceptibility to proteolysis, reducedimmunogenicity, reduced rate of absorption when administeredsubcutaneously or intramuscularly, and enhanced pharmacokineticproperties such as longer terminal half-life and increased area underthe curve (AUC), slower absorption after subcutaneous or intramuscularinjection (compared to CF not linked to XTEN and administered by asimilar route) such that the C_(max) is lower, which, in turn, resultsin reductions in adverse effects of the CF that, collectively, resultsin an increased period of time that a fusion protein of a CFXTENcomposition administered to a subject retains therapeutic activity.

A variety of methods and assays are known in the art for determining thephysical/chemical properties of proteins such as the compositionscomprising the inventive XTEN. Such properties inlcude but are notlimited to secondary or tertiary structure, solubility, proteinaggregation, melting properties, contamination and water content. Suchmethods include analytical centrifugation, EPR, HPLC-ion exchange,HPLC-size exclusion, HPLC-reverse phase, light scattering, capillaryelectrophoresis, circular dichroism, differential scanning calorimetry,fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Ramanspectroscopy, refractometry, and UV/Visible spectroscopy. Additionalmethods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48,1-13.

In one embodiment, XTEN is designed to behave like denatured peptidesequence under physiological conditions, despite the extended length ofthe polymer. “Denatured” describes the state of a peptide in solutionthat is characterized by a large conformational freedom of the peptidebackbone. Most peptides and proteins adopt a denatured conformation inthe presence of high concentrations of denaturants or at elevatedtemperature. Peptides in denatured conformation have, for example,characteristic circular dichroism (CD) spectra and are characterized bya lack of long-range interactions as determined by NMR. “Denaturedconformation” and “unstructured conformation” are used synonymouslyherein. In some embodiments, the invention provides XTEN sequences that,under physiologic conditions, resemble denatured sequences that arelargely devoid in secondary structure. In other cases, the XTENsequences are substantially devoid of secondary structure underphysiologic conditions. “Largely devoid,” as used in this context, meansthat less than 50% of the XTEN amino acid residues of the XTEN sequencecontribute to secondary structure as measured or determined by the meansdescribed herein. “Substantially devoid,” as used in this context, meansthat at least about 60%, or about 70%, or about 80%, or about 90%, orabout 95%, or at least about 99% of the XTEN amino acid residues of theXTEN sequence do not contribute to secondary structure, as measured ordetermined by the methods described herein.

A variety of methods have been established in the art to discern thepresence or absence of secondary and tertiary structures in a givenpolypeptide. In particular, secondary structure can be measuredspectrophotometrically, e.g., by circular dichroism spectroscopy in the“far-UV” spectral region (190-250 nm). Secondary structure elements,such as alpha-helix and beta-sheet, each give rise to a characteristicshape and magnitude of CD spectra. Secondary structure can also bepredicted for a polypeptide sequence via certain computer programs oralgorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y.,et al. (1974) Biochemistry, 13: 222-45) and theGarnier-Osguthorpe-Robson (“GOR”) algorithm (Gamier J, Gibrat J F,Robson B. (1996), GOR method for predicting protein secondary structurefrom amino acid sequence. Methods Enzymol 266:540-553), as described inUS Patent Application Publication No. 20030228309A1. For a givensequence, the algorithms can predict whether there exists some or nosecondary structure at all, expressed as the total and/or percentage ofresidues of the sequence that form, for example, alpha-helices orbeta-sheets or the percentage of residues of the sequence predicted toresult in random coil formation (which lacks secondary structure).

In some embodiments, the XTEN sequences used in the subject fusionprotein compositions can have an alpha-helix percentage ranging from 0%to less than about 5% as determined by the Chou-Fasman algorithm. Inother cases, the XTEN sequences of the fusion protein compositions havea beta-sheet percentage ranging from 0% to less than about 5% asdetermined by the Chou-Fasman algorithm. In some embodiments, the XTENsequences of the fusion protein compositions have an alpha-helixpercentage ranging from 0% to less than about 5% and a beta-sheetpercentage ranging from 0% to less than about 5% as determined by theChou-Fasman algorithm. In some embodiments, the XTEN sequences of thefusion protein compositions have an alpha-helix percentage less thanabout 2% and a beta-sheet percentage less than about 2%. In other cases,the XTEN sequences of the fusion protein compositions have a high degreeof random coil percentage, as determined by the GOR algorithm. In someembodiments, an XTEN sequence have at least about 80%, more preferablyat least about 90%, more preferably at least about 91%, more preferablyat least about 92%, more preferably at least about 93%, more preferablyat least about 94%, more preferably at least about 95%, more preferablyat least about 96%, more preferably at least about 97%, more preferablyat least about 98%, and most preferably at least about 99% random coil,as determined by the GOR algorithm.

1. Non-Repetitive Sequences

In some embodiments, XTEN sequences of the compositions aresubstantially non-repetitive. In general, repetitive amino acidsequences have a tendency to aggregate or form higher order structures,as exemplified by natural repetitive sequences such as collagens andleucine zippers. These repetitive amino acids may also tend to formcontacts resulting in crystalline or pseudocrystaline structures. Incontrast, the low tendency of non-repetitive sequences to aggregateenables the design of long-sequence XTENs with a relatively lowfrequency of charged amino acids that would otherwise be likely toaggregate if the sequences were repetitive. Typically, the CFXTEN fusionproteins comprise XTEN sequences of greater than about 100 to about 3000amino acid residues wherein the sequences are substantiallynon-repetitive. In one embodiment, the XTEN sequences have greater thanabout 100 to about 3000 amino acid residues in which no three contiguousamino acids in the sequence are identical amino acid types unless theamino acid is serine, in which case no more than three contiguous aminoacids are serine residues. In the foregoing embodiment, the XTENsequence is “substantially non-repetitive.”

The degree of repetitiveness of a polypeptide or a gene can be measuredby computer programs or algorithms or by other means known in the art.Repetitiveness in a polypeptide sequence can, for example, be assessedby determining the number of times shorter sequences of a given lengthoccur within the polypeptide. For example, a polypeptide of 200 aminoacid residues has 192 overlapping 9-amino acid sequences (or 9-mer“frames”) and 198 3-mer frames, but the number of unique 9-mer or 3-mersequences will depend on the amount of repetitiveness within thesequence. A score is generated (hereinafter “subsequence score”) that isreflective of the degree of repetitiveness of the subsequences in theoverall polypeptide sequence. In the context of the present invention,“subsequence score” means the sum of occurrences of each unique 3-merframe across a 200 consecutive amino acid sequence of the polypeptidedivided by the absolute number of unique 3-mer subsequences within the200 amino acid sequence. Examples of such subsequence scores derivedfrom the first 200 amino acids of repetitive and non-repetitivepolypeptides are presented in Example 44. In some embodiments, thepresent invention provides CFXTEN each comprising one or more XTEN inwhich the XTEN has a subsequence score less than 12, more preferablyless than 10, more preferably less than 9, more preferably less than 8,more preferably less than 7, more preferably less than 6, and mostpreferably less than 5. In the embodiments hereinabove described in thisparagraph, an XTEN with a subsequence score less than about 10 (i.e., 9,8, 7, etc.) is “substantially non-repetitive.”

The non-repetitive characteristic of XTEN imparts a CF fusion proteins agreater degree of solubility and less tendency to aggregate compared topolypeptides having repetitive sequences. These properties facilitatethe formulation of XTEN-comprising pharmaceutical preparationscontaining extremely high drug concentrations, in some cases exceeding100 mg/ml.

Furthermore, the XTEN polypeptide sequences of the embodiments aredesigned to have a low degree of internal repetitiveness in order toreduce or substantially eliminate immunogenicity when administered to amammal. Polypeptide sequences composed of short, repeated motifs largelylimited to three amino acids, such as glycine, serine and glutamate, mayresult in relatively high antibody titers when administered to a mammaldespite the absence of predicted T-cell epitopes in these sequences.This may be caused by the repetitive nature of polypeptides, as it hasbeen shown that immunogens with repeated epitopes, including proteinaggregates, cross-linked immunogens, and repetitive carbohydrates arehighly immunogenic and can, for example, result in the cross-linking ofB-cell receptors causing B-cell activation. (Johansson, J., et al.(2007) Vaccine, 25:1676-82; Yankai, Z., et al. (2006) Biochem BiophysRes Commun, 345:1365-71; Hsu, C. T., et al. (2000) Cancer Res,60:3701-5); Bachmann M F, et al. Eur J Immunol. (1995)25(12):3445-3451).

2. Exemplary Sequence Motifs

The present invention encompasses XTEN used as fusion partners thatcomprise multiple units of shorter sequences, or motifs, in which theamino acid sequences of the motifs are non-repetitive. Thenon-repetitive criterion can be met despite the use of a “buildingblock” approach using a library of sequence motifs that are multimerizedto create the XTEN sequences. Thus, while an XTEN sequence may consistof multiple units of as few as four different types of sequence motifs,because the motifs themselves generally consist of non-repetitive aminoacid sequences, the overall XTEN sequence is rendered substantiallynon-repetitive.

In one embodiment, XTEN have a non-repetitive sequence of greater thanabout 100 to about 3000 amino acid residues wherein at least about 80%,or at least about 85%, or at least about 90%, or at least about 95%, orat least about 97%, or about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs, wherein each of the motifs has about 9to 36 amino acid residues. In other embodiments, at least about 80%, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 97%, or about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs wherein each of the motifs has 9 to 14amino acid residues. In still other embodiments, at least about 80%, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 97%, or about 100% of the XTEN sequence component consistsof non-overlapping sequence motifs wherein each of the motifs has 12amino acid residues. In these embodiments, it is preferred that thesequence motifs be composed mainly of small hydrophilic amino acids,such that the overall sequence has an unstructured, flexiblecharacteristic. Examples of amino acids that are included in XTEN are,e.g., arginine, lysine, threonine, alanine, asparagine, glutamine,aspartate, glutamate, serine, and glycine. As a result of testingvariables such as codon optimization, assembly polynucleotides encodingsequence motifs, expression of protein, charge distribution andsolubility of expressed protein, and secondary and tertiary structure,it was discovered that XTEN compositions with enhanced characteristicsmainly include glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P) residues wherein the sequences aredesigned to be substantially non-repetitive. In one embodiment, XTENsequences have predominately four to six types of amino acids selectedfrom glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)or proline (P) that are arranged in a substantially non-repetitivesequence that is greater than about 100 to about 3000 amino acidresidues, preferably greater than 400 to about 3000 residues in length.In some embodiments, XTEN have sequences of greater than about 100 toabout 3000 amino acid residues wherein at least about 80% of thesequence consists of non-overlapping sequence motifs wherein each of themotifs has 9 to 36 amino acid residues wherein each of the motifsconsists of 4 to 6 types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),and wherein the content of any one amino acid type in the full-lengthXTEN does not exceed 30%. In other embodiments, at least about 90% ofthe XTEN sequence consists of non-overlapping sequence motifs whereineach of the motifs has 9 to 36 amino acid residues wherein the motifsconsist of 4 to 6 types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),and wherein the content of any one amino acid type in the full-lengthXTEN does not exceed 30%. In other embodiments, at least about 90% ofthe XTEN sequence consists of non-overlapping sequence motifs whereineach of the motifs has 12 amino acid residues consisting of 4 to 6 typesof amino acids selected from glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P), and wherein the content ofany one amino acid type in the full-length XTEN does not exceed 30%. Inyet other embodiments, at least about 90%, or about 91%, or about 92%,or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99%, to about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs wherein each of the motifs has 12 aminoacid residues consisting of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P), and wherein the content ofany one amino acid type in the full-length XTEN does not exceed 30%.

In still other embodiments, XTENs comprise non-repetitive sequences ofgreater than about 100 to about 3000 amino acid residues wherein atleast about 80%, or at least about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of the sequence consists of non-overlappingsequence motifs of 9 to 14 amino acid residues wherein the motifsconsist of 4 to 6 types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),and wherein the sequence of any two contiguous amino acid residues inany one motif is not repeated more than twice in the sequence motif. Inother embodiments, at least about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of an XTEN sequence consists of non-overlappingsequence motifs of 12 amino acid residues wherein the motifs consist of4 to 6 types of amino acids selected from glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P), and whereinthe sequence of any two contiguous amino acid residues in any onesequence motif is not repeated more than twice in the sequence motif. Inother embodiments, at least about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of an XTEN sequence consists of non-overlappingsequence motifs of 12 amino acid residues wherein the motifs consist ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P), and wherein the sequence of any two contiguous amino acidresidues in any one sequence motif is not repeated more than twice inthe sequence motif. In yet other embodiments, XTENs consist of 12 aminoacid sequence motifs wherein the amino acids are selected from glycine(G), alanine (A), serine (S), threonine (T), glutamate (E) and proline(P), and wherein the sequence of any two contiguous amino acid residuesin any one sequence motif is not repeated more than twice in thesequence motif, and wherein the content of any one amino acid type inthe full-length XTEN does not exceed 30%. In the foregoing embodimentshereinabove described in this paragraph, the XTEN sequences issubstantially non-repetitive.

In some embodiments, the invention provides compositions comprisingnon-repetitive XTEN sequence(s) of greater than about 100 to about 3000amino acid residues wherein at least about 80%, or at least about 90%,or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% to about 100% of thesequence consists of multiple units of two or more non-overlappingsequence motifs selected from the amino acid sequences of Table 3. Insome embodiments, the XTEN comprises non-overlapping sequence motifs inwhich about 80%, or at least about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% to about 100% of the sequence consists of two ormore non-overlapping sequences selected from a single motif family ofTable 3, resulting in a “family” sequence in which the overall sequenceremains substantially non-repetitive. Accordingly, in these embodiments,an XTEN sequence comprises multiple units of non-overlapping sequencemotifs of the AD motif family, or the AE motif family, or the AF motiffamily, or the AG motif family, or the AM motif family, or the AQ motiffamily, or the BC family, or the BD family of sequences of Table 3. Inother embodiments, the XTEN comprises motif sequences from two or moreof the motif families of Table 3.

TABLE 3 XTEN Sequence Motifs of 12 Amino Acids and Motif FamiliesMotif Family* SEQ ID NO: MOTIF SEQUENCE AD 31 GESPGGSSGSES AD 32GSEGSSGPGESS AD 33 GSSESGSSEGGP AD 34 GSGGEPSESGSS AE, AM 35GSPAGSPTSTEE AE, AM, AQ 36 GSEPATSGSETP AE, AM, AQ 37 GTSESATPESGPAE, AM, AQ 38 GTSTEPSEGSAP AF, AM 39 GSTSESPSGTAP AF, AM 40 GTSTPESGSASPAF, AM 41 GTSPSGESSTAP AF, AM 42 GSTSSTAESPGP AG, AM 43 GTPGSGTASSSPAG, AM 44 GSSTPSGATGSP AG, AM 45 GSSPSASTGTGP AG, AM 46 GASPGTSSTGSP AQ47 GEPAGSPTSTSE AQ 48 GTGEPSSTPASE AQ 49 GSGPSTESAPTE AQ 50 GSETPSGPSETAAQ 51 GPSETSTSEPGA AQ 52 GSPSEPTEGTSA BC 53 GSGASEPTSTEP BC 54GSEPATSGTEPS BC 55 GTSEPSTSEPGA BC 56 GTSTEPSEPGSA BD 57 GSTAGSETSTEA BD58 GSETATSGSETA BD 59 GTSESATSESGA BD 60 GTSTEASEGSAS *Denotesindividual motif sequences that, when used together in variouspermutations, results in a “family sequence” 

In other embodiments, the CFXTEN composition comprises a non-repetitiveXTEN sequence of greater than about 100 to about 3000 amino acidresidues, wherein at least about 80%, or at least about 90%, or about91%, or about 92%, or about 93%, or about 94%, or about 95%, or about96%, or about 97%, or about 98%, or about 99% to about 100% of thesequence consists of non-overlapping 36 amino acid sequence motifsselected from one or more of the polypeptide sequences of Tables 9-12.

In those embodiments wherein the XTEN component of the CFXTEN fusionprotein has less than 100% of its amino acids consisting of four to sixamino acid selected from glycine (G), alanine (A), serine (S), threonine(T), glutamate (E) and proline (P), or less than 100% of the sequenceconsisting of the sequence motifs of Table 3, or less than 100% sequenceidentity compared with an XTEN from Table 3, the other amino acidresidues are selected from any other of the 14 natural L-amino acids,but are preferentially selected from hydrophilic amino acids such thatthe XTEN sequence contains at least about 90%, or at least about 91%, orat least about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% hydrophilic amino acids. The XTENamino acids that are not glycine (G), alanine (A), serine (S), threonine(T), glutamate (E) and proline (P) are interspersed throughout the XTENsequence, are located within or between the sequence motifs, or areconcentrated in one or more short stretches of the XTEN sequence. Insuch cases where the XTEN component of the CFXTEN comprises amino acidsother than glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P), it is preferred that the amino acids notbe hydrophobic residues and should not substantially confer secondarystructure of the XTEN component. Hydrophobic residues that are lessfavored in construction of XTEN include tryptophan, phenylalanine,tyrosine, leucine, isoleucine, valine, and methionine. Additionally, onecan design the XTEN sequences to contain few (e.g. less than 5%) or noneof the following amino acids: cysteine (to avoid disulfide formation andoxidation), methionine (to avoid oxidation), asparagine and glutamine(to avoid desamidation). Thus, in some embodiments, the XTEN componentof the CFXTEN fusion protein comprising other amino acids in addition toglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P) would have a sequence with less than 5% of the residuescontributing to alpha-helices and beta-sheets as measured by theChou-Fasman algorithm and have at least 90%, or at least about 95% ormore random coil formation as measured by the GOR algorithm.

3. Length of Sequence

In another aspect of the present invention, the invention encompassesCFXTEN compositions comprising carriers of XTEN polypeptides withextended length sequences. The present invention makes use of thediscovery that increasing the length of the non-repetitive, unstructuredpolypeptides enhances the unstructured nature of the XTENs andcorrespondingly enhances the biological and pharmacokinetic propertiesof fusion proteins comprising the XTEN carrier. As described more fullyin the Examples, proportional increases in the length of the XTEN, evenif created by a fixed repeat order of single family sequence motifs(e.g., the four AE motifs of Table 3), result in a sequence with ahigher percentage of random coil formation, as determined by GORalgorithm, compared to shorter XTEN lengths. In general, increasing thelength of the unstructured polypeptide fusion partner, as described inthe Examples, results in a fusion protein with a disproportionateincrease in terminal half-life compared to fusion proteins withunstructured polypeptide partners with shorter sequence lengths.

Non-limiting examples of XTEN contemplated for inclusion in the CFXTENof the invention are presented in Table 4, below. In one embodiment, theinvention provides CFXTEN compositions wherein the XTEN sequence lengthof the fusion protein(s) is greater than about 100 to about 3000 aminoacid residues, and in some cases is greater than 400 to about 3000 aminoacid residues, wherein the XTEN confers enhanced pharmacokineticproperties on the CFXTEN in comparison to CF not linked to XTEN. In someembodiments, the XTEN sequences of the CFXTEN compositions of thepresent invention can be about 100, or about 144, or about 288, or about401, or about 500, or about 600, or about 700, or about 800, or about900, or about 1000, or about 1500, or about 2000, or about 2500 or up toabout 3000 amino acid residues in length. In other cases, the XTENsequences can be about 100 to 150, about 150 to 250, about 250 to 400,401 to about 500, about 500 to 900, about 900 to 1500, about 1500 to2000, or about 2000 to about 3000 amino acid residues in length. In oneembodiment, the CFXTEN can comprise an XTEN sequence wherein thesequence exhibits at least about 80% sequence identity, or alternatively81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a XTENselected from Table 4. In some embodiments, the XTEN sequence isdesigned for optimized expression as the N-terminal component of theCFXTEN by inclusion of encoding nucleotides for an optimized N-terminalleader sequence (NTS) in the XTEN portion of the gene encoding thefusion protein. In one embodiment, the N-terminal XTEN sequence of theexpressed CFXTEN has at least 90% sequence identity compared to thesequence of AE48 or AM48, AE624, or AE912 or AM923. In anotherembodiment, the XTEN has the N-terminal residues described in Examples14-17.

In other embodiments, the CFXTEN fusion protein comprises a first and asecond XTEN sequence, wherein the cumulative total of the residues inthe XTEN sequences is greater than about 400 to about 3000 amino acidresidues and the XTEN can be identical or they can be different insequence. In embodiments of the foregoing, the CFXTEN fusion proteincomprises a first and a second XTEN sequence wherein the sequences eachexhibit at least about 80% sequence identity, or alternatively 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity compared to at least a first oradditionally a second XTEN selected from Table 4. Examples where morethan one XTEN is used in a CFXTEN composition include, but are notlimited to constructs with an XTEN linked to both the N- and C-terminiof at least one CF.

As described more fully below, the invention provides methods in whichthe CFXTEN is designed by selecting the length of the XTEN to confer atarget half-life on a fusion protein administered to a subject. Ingeneral, XTEN lengths longer that about cumulative 400 residuesincorporated into the CFXTEN compositions result in longer half-lifecompared to shorter cumulative lengths; e.g., shorter than about 280residues. However, in another embodiment, CFXTEN fusion proteins aredesigned to comprise XTEN with a longer sequence length that is selectedto additionally confer slower rates of systemic absorption aftersubcutaneous or intramuscular administration to a subject. In suchembodiments, the C_(max) is reduced in comparison to a comparable doseof a CF not linked to XTEN, thereby contributing to the ability to keepthe CFXTEN within the therapeutic window for the composition. Thus, theXTEN confers the property of a depot to the administered CFXTEN, inaddition to the other physical/chemical properties described herein.

TABLE 4 XTEN Polypeptides XTEN SEQ ID Name NO: Amino Acid Sequence AE4861 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS AM48 62MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS AE144 63GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AF144 64GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAP AE288 65GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AF504 66GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AF540 67GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AD576 68GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSS GPGESS AE57669 GSPAGSPTSTEEGTSESATPESGPGTSFEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AF576 70GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP AE624 71MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSFEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSFEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AD836 72GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS AE864 73GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AF864 74GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTP SGATGSP AG86475 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPAM875 76 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPAE912 77 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AM923 78MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAP AM131879 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP BC864 80GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA BD864 81GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA Y288 82GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGE GSEGSGEGEGSEGSGEY576 83 GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEGSEGEGSEGSGEGEGSGEGSE

4. XTEN Segments

In one embodiment, the invention provides an isolated CFXTEN fusionprotein wherein the cumulative length of the XTEN component is greaterthan about 100 to about 3000 amino acid residues containing at least onepolypeptide sequence segment selected from Tables 4, 9, 10, 11, 12, and13 and wherein at least about 90%, or at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98% or more of the remainder of the XTEN sequence containshydrophilic amino acids and less than about 2% of the remainder of theXTEN consists of hydrophobic or aromatic amino acids or cysteine. Insome embodiments, the XTEN contains multiple segments wherein thesegments are identical or different. In another embodiment, theinvention provides an isolated CFXTEN fusion protein wherein thecumulative length of the XTEN component is greater than about 100 toabout 3000 amino acid residues and comprises at least one sequencesegment of at least about 100 to about 923, or at least about 100 toabout 875, or at least about 100 to about 576, or at least about 100 toabout 288, or at least about 100 to about 144 amino acid residueswherein the sequence segment(s) consists of at least three differenttypes of amino acids and the sum of glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) and proline (P) residues in thesequence segment(s) constitutes at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% of the total aminoacid sequence of the sequence segment and at least about 90%, or atleast about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98% of the remainder of the XTENsequence(s) consist of hydrophilic amino acids and less than about 2% ofthe remainder of the XTEN sequence(s) consists of hydrophobic oraromatic amino acids, or cysteine. In another embodiment, the inventionprovides an isolated CFXTEN fusion protein wherein the cumulative lengthof the XTEN component is greater than about 100 to about 3000 amino acidresidues and comprises at least one sequence segment of at least about200 to about 923, or at least about 200 to about 875, or at least about200 to about 576, or at least about 200 to about 288 amino acid residueswherein the sequence segment(s) the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P) residues in thesequence segment(s) constitutes at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% of the total aminoacid sequence of the sequence segment and wherein the subsequence scoreof the segment is less than 12, more preferably less than 10, morepreferably less than 9, more preferably less than 8, more preferablyless than 7, more preferably less than 6, and most preferably less than5, and at least about 90%, or at least about 91%, or at least about 92%,or at least about 93%, or at least about 94%, or at least about 95%, orat least about 96%, or at least about 97%, or at least about 98% of theremainder of the XTEN sequence(s) consist of hydrophilic amino acids andless than about 2% of the remainder of the XTEN sequence(s) consists ofhydrophobic, aromatic or cysteine amino acids.

5. N-Terminal XTEN Expression-Enhancing Sequences

In some embodiments, the invention provides a short-length XTEN sequenceincorporated as the N-terminal portion of the CFXTEN fusion protein. Ithas been discovered that the expression of the fusion protein isenhanced in a host cell transformed with a suitable expression vectorcomprising an optimized N-terminal leader polynucleotide sequence (thatencodes the N-terminal XTEN) incorporated into the polynucleotideencoding the binding fusion protein. As described in Examples 14-17, ahost cell transformed with such an expression vector comprising anoptimized N-terminal leader sequence (NTS) in the binding fusion proteingene results in greatly-enhanced expression of the fusion proteincompared to the expression of a corresponding fusion protein from apolynucleotide not comprising the NTS, and obviates the need forincorporation of a non-XTEN leader sequence used to enhance expression.In one embodiment, the invention provides CFXTEN fusion proteinscomprising an NTS wherein the expression of the binding fusion proteinfrom the encoding gene in a host cell is enhanced about 50%, or about75%, or about 100%, or about 150%, or about 200%, or about 400% comparedto expression of a CFXTEN fusion protein not comprising the N-terminalXTEN sequence (where the encoding gene lacks the NTS).

In one embodiment, the N-terminal XTEN polypeptide of the CFXTENcomprises a sequence that exhibits at least about 80%, more preferablyat least about 90%, more preferably at least about 91%, more preferablyat least about 92%, more preferably at least about 93%, more preferablyat least about 94%, more preferably at least about 95%, more preferablyat least about 96%, more preferably at least about 97%, more preferablyat least about 98%, more preferably at least 99%, or exhibits 100%sequence identity compared to the amino acid sequence of AE48 or AM48,the respective amino acid sequences of which are as follows:

AE48: (SEQ ID NO: 61) MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSAM48: (SEQ ID NO: 62) MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS

In another embodiment, the short-length N-terminal XTEN is linked to anXTEN of longer length to form the N-terminal region of the CFXTEN fusionprotein, wherein the polynucleotide sequence encoding the short-lengthN-terminal XTEN confers the property of enhanced expression in the hostcell, and wherein the long length of the expressed XTEN contributes tothe enhanced properties of the XTEN carrier in the fusion protein, asdescribed above. In the foregoing, the short-length XTEN is linked toany of the XTEN disclosed herein (e.g., an XTEN of Table 3) and theresulting XTEN, in turn, is linked to the N-terminal of any of the CFdisclosed herein (e.g., a CF of Table 1 or Table 2) as a component ofthe fusion protein. Alternatively, polynucleotides encoding theshort-length XTEN (or its complement) is linked to polynucleotidesencoding any of the XTEN (or its complement) disclosed herein and theresulting gene encoding the N-terminal XTEN, in turn, is linked to the5′ end of polynucleotides encoding any of the CF (or to the 3′ end ofits complement) disclosed herein. In some embodiments, the N-terminalXTEN polypeptide with long length exhibits at least about 80%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast 99%, or exhibits 100% sequence identity compared to an amino acidsequence selected from the group consisting of the sequences AE624,AE912, and AM923.

In any of the foregoing N-terminal XTEN embodiments described above, theN-terminal XTEN can have from about one to about six additional aminoacid residues, preferably selected from GESTPA, to accommodate therestriction endonuclease restriction sites that is employed to join thenucleotides encoding the N-terminal XTEN to the gene encoding thetargeting moiety of the fusion protein. The methods for the generationof the N-terminal sequences and incorporation into the fusion proteinsof the invention are described more fully in the Examples.

6. Net Charge

In other embodiments, the XTEN polypeptides have an unstructuredcharacteristic imparted by incorporation of amino acid residues with anet charge and/or reducing the proportion of hydrophobic amino acids inthe XTEN sequence. The overall net charge and net charge density iscontrolled by modifying the content of charged amino acids in the XTENsequences. In some embodiments, the net charge density of the XTEN ofthe compositions may be above +0.1 or below −0.1 charges/residue. By“net charge density” of a protein or peptide herein is meant the netcharge divided by the total number of amino acids in the protein orproptide. In other embodiments, the net charge density of a XTEN can beabout 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, orabout 20% or more.

Since most tissues and surfaces in a human or animal have a net negativecharge, in some embodiments, the XTEN sequences are designed to have anet negative charge to minimize non-specific interactions between theXTEN containing compositions and various surfaces such as blood vessels,healthy tissues, or various receptors. Not to be bound by a particulartheory, the XTEN can adopt open conformations due to electrostaticrepulsion between individual amino acids of the XTEN polypeptide thatindividually carry a net negative charge and that are distributed acrossthe sequence of the XTEN polypeptide. Such a distribution of netnegative charge in the extended sequence lengths of XTEN can lead to anunstructured conformation that, in turn, can result in an effectiveincrease in hydrodynamic radius. In preferred embodiments, the negativecharge is conferred by incorporation of glutamic acid residues.Accordingly, in one embodiment the invention provides XTEN in which theXTEN sequences contain about 8, 10, 15, 20, 25, or even about 30%glutamic acid. Generally, the glutamic residues is spaced uniformlyacross the XTEN sequence. In some cases, the XTEN can contain about10-80, or about 15-60, or about 20-50 glutamic residues per 20 kDa ofXTEN that can result in an XTEN with charged residues that would havevery similar pKa, which can increase the charge homogeneity of theproduct and sharpen its isoelectric point, enhance the physicochemicalproperties of the resulting CFXTEN fusion protein for, and hence,simplifying purification procedures.

The XTEN of the compositions of the present invention generally have noor a low content of positively charged amino acids. In some embodiments,the XTEN may have less than about 10% amino acid residues with apositive charge, or less than about 7%, or less than about 5%, or lessthan about 2%, or less than about 1% amino acid residues with a positivecharge. However, the invention contemplates constructs where a limitednumber of amino acids with a positive charge, such as lysine, areincorporated into XTEN to permit conjugation between the epsilon amineof the lysine and a reactive group on a peptide, a linker bridge, or areactive group on a drug or small molecule to be conjugated to the XTENbackbone. In one embodiment of the foregoing, the XTEN has between about1 to about 100 lysine residues, or about 1 to about 70 lysine residues,or about 1 to about 50 lysine residues, or about 1 to about 30 lysineresidues, or about 1 to about 20 lysine residues, or about 1 to about 10lysine residues, or about 1 to about 5 lysine residues, or alternativelyonly a single lysine residue. Using the foregoing lysine-containingXTEN, fusion proteins are constructed that comprises XTEN, a coagulationfactor, plus a chemotherapeutic agent useful in the treatment ofgrowth-related diseases or disorders, wherein the maximum number ofmolecules of the agent incorporated into the XTEN component isdetermined by the numbers of lysines or other amino acids with reactiveside chains (e.g., cysteine) incorporated into the XTEN.

In some embodiments, the XTEN sequence comprises charged residuesseparated by other residues such as serine or glycine, which leads tobetter expression or purification behavior. Based on the net charge,some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, theXTEN will have an isoelectric point between 1.5 and 4.5. In theseembodiments, the XTEN incorporated into the CFXTEN fusion proteincompositions of the present invention carry a net negative charge underphysiologic conditions that contribute to the unstructured conformationand reduced binding of the XTEN component to mammalian proteins andtissues.

As hydrophobic amino acids impart structure to a polypeptide, theinvention provides that the content of hydrophobic amino acids in theXTEN will typically be less than 5%, or less than 2%, or less than 1%hydrophobic amino acid content. In one embodiment, the amino acidcontent of methionine and tryptophan in the XTEN component of a CFXTENfusion protein is typically less than 5%, or less than 2%, and mostpreferably less than 1%. In another embodiment, the XTEN will have asequence that has less than 10% amino acid residues with a positivecharge, or less than about 7%, or less that about 5%, or less than about2% amino acid residues with a positive charge, the sum of methionine andtryptophan residues will be less than 2%, and the sum of asparagine andglutamine residues will be less than 10% of the total XTEN sequence.

7. Low Immunogenicity

In another aspect, the invention provides compositions in which the XTENsequences have a low degree of immunogenicity or are substantiallynon-immunogenic. Several factors can contribute to the lowimmunogenicity of XTEN, e.g., the non-repetitive sequence, theunstructured conformation, the high degree of solubility, the low degreeor lack of self-aggregation, the low degree or lack of proteolytic siteswithin the sequence, and the low degree or lack of epitopes in the XTENsequence.

Conformational epitopes are formed by regions of the protein surfacethat are composed of multiple discontinuous amino acid sequences of theprotein antigen. The precise folding of the protein brings thesesequences into a well-defined, stable spatial configurations, orepitopes, that can be recognized as “foreign” by the host humoral immunesystem, resulting in the production of antibodies to the protein or theactivation of a cell-mediated immune response. In the latter case, theimmune response to a protein in an individual is heavily influenced byT-cell epitope recognition that is a function of the peptide bindingspecificity of that individual's HLA-DR allotype. Engagement of a MHCClass II peptide complex by a cognate T-cell receptor on the surface ofthe T-cell, together with the cross-binding of certain otherco-receptors such as the CD4 molecule, can induce an activated statewithin the T-cell. Activation leads to the release of cytokines furtheractivating other lymphocytes such as B cells to produce antibodies oractivating T killer cells as a full cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule forpresentation on the surface of an APC (antigen presenting cell) isdependent on a number of factors; most notably its primary sequence. Inone embodiment, a lower degree of immunogenicity is achieved bydesigning XTEN sequences that resist antigen processing in antigenpresenting cells, and/or choosing sequences that do not bind MHCreceptors well. The invention provides CFXTEN fusion proteins withsubstantially non-repetitive XTEN polypeptides designed to reducebinding with MHC II receptors, as well as avoiding formation of epitopesfor T-cell receptor or antibody binding, resulting in a low degree ofimmunogenicity. Avoidance of immunogenicity can attribute to, at leastin part, a result of the conformational flexibility of XTEN sequences;i.e., the lack of secondary structure due to the selection and order ofamino acid residues. For example, of particular interest are sequenceshaving a low tendency to adapt compactly folded conformations in aqueoussolution or under physiologic conditions that could result inconformational epitopes. The administration of fusion proteinscomprising XTEN, using conventional therapeutic practices and dosing,would generally not result in the formation of neutralizing antibodiesto the XTEN sequence, and also reduce the immunogenicity of the CFfusion partner in the CFXTEN compositions.

In one embodiment, the XTEN sequences utilized in the subject fusionproteins can be substantially free of epitopes recognized by human Tcells. The elimination of such epitopes for the purpose of generatingless immunogenic proteins has been disclosed previously; see for exampleWO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated byreference herein. Assays for human T cell epitopes have been described(Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Ofparticular interest are peptide sequences that can be oligomerizedwithout generating T cell epitopes or non-human sequences. This isachieved by testing direct repeats of these sequences for the presenceof T-cell epitopes and for the occurrence of 6 to 15-mer and, inparticular, 9-mer sequences that are not human, and then altering thedesign of the XTEN sequence to eliminate or disrupt the epitopesequence. In some embodiments, the XTEN sequences are substantiallynon-immunogenic by the restriction of the numbers of epitopes of theXTEN predicted to bind MHC receptors. With a reduction in the numbers ofepitopes capable of binding to MHC receptors, there is a concomitantreduction in the potential for T cell activation as well as T cellhelper function, reduced B cell activation or upregulation and reducedantibody production. The low degree of predicted T-cell epitopes can bedetermined by epitope prediction algorithms such as, e.g., TEPITOPE(Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown inExample 45. The TEPITOPE score of a given peptide frame within a proteinis the log of the K_(d) (dissociation constant, affinity, off-rate) ofthe binding of that peptide frame to multiple of the most common humanMHC alleles, as disclosed in Sturniolo, T. et al. (1999) NatureBiotechnology 17:555). The score ranges over at least 20 logs, fromabout 10 to about −10 (corresponding to binding constraints of 10e¹⁰K_(d) to 10e⁻¹⁰ K_(d)), and can be reduced by avoiding hydrophobic aminoacids that serve as anchor residues during peptide display on MHC, suchas M, I, L, V, F. In some embodiments, an XTEN component incorporatedinto a CFXTEN does not have a predicted T-cell epitope at a TEPITOPEscore of about −5 or greater, or −6 or greater, or −7 or greater, or −8or greater, or at a TEPITOPE score of −9 or greater. As used herein, ascore of “−9 or greater” would encompass TEPITOPE scores of 10 to −9,inclusive, but would not encompass a score of −10, as −10 is less than−9.

In another embodiment, the inventive XTEN sequences, including thoseincorporated into the subject CFXTEN fusion proteins, are renderedsubstantially non-immunogenic by the restriction of known proteolyticsites from the sequence of the XTEN, reducing the processing of XTENinto small peptides that can bind to MHC II receptors. In anotherembodiment, the XTEN sequence is rendered substantially non-immunogenicby the use a sequence that is substantially devoid of secondarystructure, conferring resistance to many proteases due to the highentropy of the structure. Accordingly, the reduced TEPITOPE score andelimination of known proteolytic sites from the XTEN render the XTENcompositions, including the XTEN of the CFXTEN fusion proteincompositions, substantially unable to be bound by mammalian receptors,including those of the immune system. In one embodiment, an XTEN of aCFXTEN fusion protein can have >100 nM K_(d) binding to a mammalianreceptor, or greater than 500 nM K_(d), or greater than 1 μM K_(d)towards a mammalian cell surface or circulating polypeptide receptor.

Additionally, the non-repetitive sequence and corresponding lack ofepitopes of XTEN limit the ability of B cells to bind to or be activatedby XTEN. A repetitive sequence is recognized and can form multivalentcontacts with even a few B cells and, as a consequence of thecross-linking of multiple T-cell independent receptors, can stimulate Bcell proliferation and antibody production. In contrast, while a XTENcan make contacts with many different B cells over its extendedsequence, each individual B cell may only make one or a small number ofcontacts with an individual XTEN due to the lack of repetitiveness ofthe sequence. Not being to be bound by any theory, XTENs typically havea much lower tendency to stimulate proliferation of B cells and thus animmune response. In one embodiment, the CFXTEN have reducedimmunogenicity as compared to the corresponding CF that is not fused toan XTENT. In one embodiment, the administration of up to threeparenteral doses of a CFXTEN to a mammal result in detectableanti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution of1:1000. In another embodiment, the administration of up to threeparenteral doses of a CFXTEN to a mammal result in detectable anti-CFIgG at a serum dilution of 1:100 but not at a dilution of 1:1000. Inanother embodiment, the administration of up to three parenteral dosesof a CFXTEN to a mammal result in detectable anti-XTEN IgG at a serumdilution of 1:100 but not at a dilution of 1:1000. In the foregoingembodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgusmonkey.

An additional feature of XTENs with non-repetitive sequences relative tosequences with a high degree of repetitiveness is non-repetitive XTENsform weaker contacts with antibodies. Antibodies are multivalentmolecules. For instance, IgGs have two identical binding sites and IgMscontain 10 identical binding sites. Thus antibodies against repetitivesequences can form multivalent contacts with such repetitive sequenceswith high avidity, which can affect the potency and/or elimination ofsuch repetitive sequences. In contrast, antibodies againstnon-repetitive XTENs may yield monovalent interactions, resulting inless likelihood of immune clearance such that the CFXTEN compositionscan remain in circulation for an increased period of time.

8. Increased Hydrodynamic Radius

In another aspect, the present invention provides XTEN in which the XTENpolypeptides have a high hydrodynamic radius that confers acorresponding increased apparent molecular weight to the CFXTEN fusionprotein incorporating the XTEN. As detailed in Example 38, the linkingof XTEN to CF sequences, such as FIX or FVII sequences, results inCFXTEN compositions that can have increased hydrodynamic radii,increased apparent molecular weight, and increased apparent molecularweight factor compared to a CF not linked to an XTEN. For example, intherapeutic applications in which prolonged half-life is desired,compositions in which a XTEN with a high hydrodynamic radius isincorporated into a fusion protein comprising CF can effectively enlargethe hydrodynamic radius of the composition beyond the glomerular poresize of approximately 3-5 nm (corresponding to an apparent molecularweight of about 70 kDA, which is larger than both native FIX and FVII)(Caliceti. 2003. Pharmacokinetic and biodistribution properties ofpoly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev55:1261-1277), resulting in reduced renal clearance of circulatingproteins. The hydrodynamic radius of a protein is determined by itsmolecular weight as well as by its structure, including shape orcompactness. Not to be bound by a particular theory, the XTEN can adoptopen conformations due to electrostatic repulsion between individualcharges of the peptide or the inherent flexibility imparted by theparticular amino acids in the sequence that lack potential to confersecondary structure. The open, extended and unstructured conformation ofthe XTEN polypeptide can have a greater proportional hydrodynamic radiuscompared to polypeptides of a comparable sequence length and/ormolecular weight that have secondary and/or tertiary structure, such astypical globular proteins. Methods for determining the hydrodynamicradius are well known in the art, such as by the use of size exclusionchromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and7,294,513. As the results of Example 38 demonstrate, the addition ofincreasing lengths of XTEN results in proportional increases in theparameters of hydrodynamic radius, apparent molecular weight, andapparent molecular weight factor, permitting the tailoring of CFXTEN todesired characteristic cut-off apparent molecular weights orhydrodynamic radii. Accordingly, in certain embodiments, the CFXTENfusion protein can be configured with an XTEN such that the fusionprotein can have a hydrodynamic radius of at least about 5 nm, or atleast about 8 nm, or at least about 10 nm, or 12 nm, or at least about15 nm. In the foregoing embodiments, the large hydrodynamic radiusconferred by the XTEN in an CFXTEN fusion protein can lead to reducedrenal clearance of the resulting fusion protein, leading to acorresponding increase in terminal half-life, an increase in meanresidence time, and/or a decrease in renal clearance rate.

In another embodiment, an XTEN of a chosen length and sequence (e.g., asequence from Table 4 or a sequence variant thereof) can be selectivelyincorporated into a CFXTEN to create a fusion protein that have, underphysiologic conditions, an apparent molecular weight of at least about500 kDa, or at least about 800 kDa, or at least about 1000 kDa, or atleast about 1500 kDA, or at least about 1800 kDa, or at least about 2000kDa, or at least about 2300 kDa or more. In another embodiment, an XTENof a chosen length and sequence can be selectively linked to a CF toresult in a CFXTEN fusion protein that has, under physiologicconditions, an apparent molecular weight factor of at least four,alternatively of at least five, alternatively of at least six,alternatively of at least eight, alternatively of at least 10,alternatively of at least 15, or an apparent molecular weight factor ofat least 20 or greater. In another embodiment, the CFXTEN fusion proteinhas, under physiologic conditions, an apparent molecular weight factorthat is about 4 to about 20, or is about 6 to about 15, or is about 8 toabout 12, or is about 9 to about 10 relative to the actual molecularweight of the fusion protein.

V). CFXTEN Variants, Structural Configurations and Properties

The CF of the subject compositions are not limited to native,full-length FIX or FVII polypeptides, but also include recombinantversions as well as biologically and/or pharmacologically active formswith sequence variants, combinations of FVII and FIX sequences, orfragments thereof. For example, it will be appreciated that variousamino acid deletions, insertions and substitutions can be made in the CFto create variants without departing from the spirit of the inventionwith respect to the biological activity or pharmacologic properties ofthe CF. Examples of conservative substitutions for amino acids inpolypeptide sequences are shown in Table 5. However, in embodiments ofthe CFXTEN in which the sequence identity of the CF is less than 100%compared to a specific sequence disclosed herein, the inventioncontemplates substitution of any of the other 19 natural L-amino acidsfor a given amino acid residue of the given CF (e.g., FIX or FVII),which may be at any position within the sequence of the CF, includingadjacent amino acid residues. If any one substitution results in anundesirable change in biological activity, then one of the alternativeamino acids can be employed and the construct evaluated by the methodsdescribed herein, or using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934, the contents of which is incorporated byreference in its entirety, or using methods generally known in the art.In addition, variants can include, for instance, polypeptides whereinone or more amino acid residues are added or deleted at the N- orC-terminus of the full-length native amino acid sequence of a CF thatretains some if not all of the biological activity of the nativepeptide; e.g., the ability to activate another coagulation factor and/orparticipate in the coagulation cascade, leading to fibrin formation andhemostasis.

In one embodiment, a factor IX incorporated into a CFXTEN fusion proteinhas a sequence that exhibits at least about 80% sequence identitycompared to a sequence from Table 1, alternatively at least about 81%,or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, orabout 87%, or about 88%, or about 89%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99%, sequence identity as comparedwith a sequence from Table 1.

In one embodiment, a factor VII incorporated into a CFXTEN fusionprotein has a sequence that exhibits at least about 80% sequenceidentity compared to a sequence from Table 2, alternatively at leastabout 81%, or about 82%, or about 83%, or about 84%, or about 85%, orabout 86%, or about 87%, or about 88%, or about 89%, or about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99%, sequence identityas compared with a sequence from Table 2.

TABLE 5 Exemplary conservative amino acid substitutions Original ResidueExemplary Substitutions Ala (A) val; leu; ile Arg (R) lys; gln; asn Asn(N) gin; his; lys; arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) AspGly (G) Pro His (H) asn: gin: lys: arg xIle (I) leu; val; met; ala; phe:norleucine Leu (L) norleucine: ile: val; met; ala: phe Lys (K) arg: gin:asn Met (M) leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) Gly Ser (S)Thr Thr (T) Ser Trp (W) Tyr Tyr(Y) Trp: phe: thr: ser Val (V) Ile; leu;met; phe; ala; norleucine

1. Internal XTEN Sequences

The present invention encompasses CFXTEN that comprise one or more XTENsequences located internal to the CF sequence. The one or moreinternally-located XTEN can be a sequence length of 36 to ≥1000 aminoacid residues. In some embodiments, the CFXTEN can have one or two orthree or four or more XTEN sequences with at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity compared to one or more XTEN selected from Tables 4, 9, 10, 11,12 and 13 wherein the XTEN sequences are located internal to the CFsequence. In one embodiment of the foregoing, the CFXTEN with one ormore internal XTEN has an additional XTEN located at the N- orC-terminus of the fusion protein with at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity compared to one or more XTEN selected from Table 4. In anotherembodiment, the invention provided CFXTEN with internal XTEN (asdetailed below) that further comprises a C-terminus XTEN linked to theCF by a cleavage sequence (e.g., a cleavage sequence of Table 7) suchthat the XTEN can be released when acted on by a protease. The linkageof XTEN by cleavage sequences is more fully described below and in theExamples.

In some embodiments, as illustrated in FIG. 2 and described more fullyin the Examples, an XTEN can be located between the domains of a FIXsequence; e.g., between the Gla and EGF1, or between the EGF1 and EGF2,or between the EGF2 and the activation peptide, or within the sequenceof the activation peptide between the R145-A146 and R180-V181 activationpeptide residues of the AP (i.e., between any two amino acids of thesequence TVFPDVDYVNSTEAETILDNITQSTQSFNDF (SEQ ID NO: 84)), or betweenthe EGF2 and the activation peptide, or between the activation peptideand the protease domain, or any combination of the foregoing. In otherembodiments, as illustrated in FIG. 2 and detailed more fully in theExamples, the XTEN can be inserted within an existing loop sequencewithin an individual domain of the FIX sequence so that 1) the XTENforms a looped structure outside the domain and doesn't disrupt thenormal architecture of the domain; and 2) the XTEN can be released bycleavage of incorporated cleavage sites.

In another embodiment, the invention provides a CFXTEN comprising a FVIIthat incorporates one or more XTEN located between the domains of a FVIIsequence; e.g., between the Gla and EGF1, or between the EGF1 and EGF2,or between the EGF2 and the activating peptide, or between theactivating peptide and the protease domain, or any combination of theforegoing. The XTEN can be a sequence of 36 to >1000 amino acid residuesincluding, but not limited to a sequence that has at least about 80%, orat least about 85%, or at least about 90%, or at least about 95% or moresequence identity compared to a sequence from Table 4, 8, 9, 10, 11, 12,and 13. In one embodiment, as illustrated in FIGS. 5 and 6, an XTEN isincorporated between the EGF2 domain and the single lytic cleavage siteat residues Arg¹⁵²-Ile¹⁵³. In other embodiments, as illustrated in FIGS.5 and 6 and detailed more fully in the Examples, the XTEN can beinserted within an existing loop sequence within an individual domain ofthe FVII sequence so that 1) the XTEN forms a looped structure outsidethe domain and doesn't disrupt the normal architecture of the domain;and 2) the XTEN can be released by cleavage of incorporated cleavagesites.

2. Factor VII-FIX Hybrid Sequence Variants

The invention provides an isolated factor VII polypeptide comprising atleast one heterologous sequence that is cleavable by a pro-coagulantprotease that does not activate a wildtype factor VII, wherein uponcleavage heterologous sequence, the factor VII polypeptide is activated.For example, CFXTEN with factor VII-factor IX hybrid sequence variantsthat incorporate into, or replace a portion of the sequence, a factorVII construct portions of the activating peptide domain (AP) sequencefrom factor IX, resulting in hybrid compositions that can be activatedas part of the intrinsic system of the coagulation cascade. The CFXTENthat incorporate such factor VII-factor IX sequence variants as the CFcomponent of the fusion protein permit administration to a subject acomposition in which the CF component is not activated, and can be dosedat high amounts because it remains as an inert, circulating depot thatis largely resistant to inactivation by protease inhibitors untilactivated by the triggering of the intrinsic coagulation cascade or byauto-activation, the latter a slow process. Non-limiting examples ofFVII/FIX hybrid sequences are illustrated in FIG. 36, showing thoseportions of the hybrid amino acid sequences that have homology withthose of native FIX and FVII. In some embodiments, the CFXTEN comprisefactor VII-factor IX sequence variants that substitute portions or theentirety of the FIX activating peptide sequence with one or both FIX APcleavage sites for FVII sequence to the N-terminal side of the proteasedomain of FVII; i.e., either towards the N-terminus beginning with thearginine at position 212 of the full-length precursor polypeptide or theisoleucine at position 213. In one embodiment, the factor VII-factor IXsequence CF incorporates the full-length FIX AP domain plus at leastabout 2, or at least about 3, or at least about 4, or at least about 5,or at least about 6, or at least about 7, or at least about 8, or atleast about 9, or at least about 10, or at least about 11, or at leastabout 12 amino acids flanking adjacent amino acid residues on one orboth sides of the R145-A146 and R180-V181 cleavage sites of FIX (e.g.,the sequence RVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE (SEQID NO: 1) in the case of 12 flanking amino acids on the N-terminus sideand 5 flanking amino acids on the C-terminus side). In anotherembodiment, the CFXTEN comprises a factor VII-factor IX sequence variantthat incorporates a portion of the AP that includes a sequence of atleast about 2, or at least about 3, or at least about 4, or at leastabout 5 that flank the R145-A146 AP cleavage site (e.g., the sequenceTSKLTRAETVFP (SEQ ID NO: 3) in the case of 6 flanking amino acids oneither side of the cleavage site). In another embodiment, the CFXTENcomprises a factor VII-factor IX sequence variant that incorporates aportion of the AP that includes a sequence of at least about 2, or atleast about 3, or at least about 4, or at least about 5 amino acids thatflank one or both sides of the R180-V181 AP cleavage site (e.g., thesequence and DFTRV (SEQ ID NO: 4) in the case of 4 amino acids on theN-terminal flank and valine as the C-terminus of the cleavage site fromFIX). In another embodiment, the CFXTEN comprises the factor VII-factorIX sequence variant of any of the foregoing embodiments of thisparagraph that further includes the same AP sequence as a linker betweenthe C-terminus of the FVII component and the XTEN component of thefusion protein; e.g., an N- to C-terminus configuration of FVIIvariant-AP sequence-XTEN, thereby permitting the release of the factorVII-factor IX sequence variant component from the CFXTEN fusion proteinby the same intrinsic coagulation factor as per that of the FVII toFVIIa transition. In another embodiment, the CFXTEN comprises the factorVII-factor IX sequence variant of any of the foregoing embodiments ofthis paragraph that further includes the factor XI cleavage sequenceKLTRAET (SEQ ID NO: 6) as the linker between the FVII variant sequenceand the XTEN, thereby permitting the release of the factor VII-factor IXsequence variant component from the CFXTEN fusion protein by theinitiation of the intrinsic coagulation cascade. It is expected d thatwith the release of the XTEN from the factor VII-factor IX sequencevariant, the activated factor VII-factor IX sequence variant would havea shorter half-life compared to the intact CFXTEN, thereby increasingthe margin of safety and tolerability of the composition in a subject.In the embodiments of the paragraph, the activated factor VII-factor IXsequence variant molecule can have at least about 60%, or at least about70%, or at least about 80%, or at least about 90%, or at least about 95%of the biological activity as native FVIIa, as measured by any of theappropriate assays or parameters disclosed herein (e.g., PT or bleedingtime assays).

In yet another embodiment, the invention provides the factor VII-factorIX sequence variants of the foregoing embodiments of this paragraphwithout a linked XTEN, permitting their administration to a subject as acirculating depot of the factor VII-factor IX hybrid that can beactivated by either the intrinsic or extrinsic coagulation cascade. Inone embodiment, the invention provides a CFXTEN with a factor VII-factorIX sequence variant with incorporated FIX-derived sequence with anoverall sequence that exhibits at least about 80% sequence identity, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%, sequence identity compared to a sequence from Table 43.In another embodiment, the invention provides a factor VII-factor IXsequence variant with incorporated FIX-derived cleavage sequence(without an XTEN) with a sequence that exhibits at least about 80%sequence identity, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99%, sequence identity as comparedwith a sequence from Table 43 without an XTEN.

The CFXTEN comprising factor VII-factor IX sequence variants can beevaluated for biological activity using assays or in vivo parameters asdescribed herein (e.g., in vitro coagulation assays or a pharmacodynamiceffect in a hemophilia model), and those sequences that retain at leastabout 40%, or about 50%, or about 55%, or about 60%, or about 70%, orabout 80%, or about 90%, or about 95% or more activity compared to thecorresponding native FVII sequence is considered suitable for inclusionin the subject CFXTEN. The CF found to retain a suitable level ofactivity can be linked to one or more XTEN polypeptides describedhereinabove. In one embodiment, a CF found to retain a suitable level ofactivity can be linked to one or more XTEN polypeptides having at leastabout 80% sequence identity to a sequence from Table 4, alternatively atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity ascompared with a sequence of Table 4, resulting in a chimeric fusionprotein.

3. CFXTEN Fusion Protein Configurations

The invention provides CFXTEN fusion protein compositions with the CFand XTEN components linked in specific N- to C-terminus configurations.In some embodiments, one or more CFs are linked to one or more XTENs,either at the N-terminus or at the C-terminus, with or without a spacer,to form a block copolymer, and the sequential arrangement of the CFs andthe XTENs in the CFXTEN fusion protein are the same as the configurationknown in the block copolymer chemistry. When there is more than one CF,XTEN, or spacer, each of the CF, the XTEN, or the spacer have the sameor different sequences, and the CFs and/or XTENs are linked eithercontinuously or alternately (regular or irregular). Thus, in all of theformulae provided herein, when there is more than one CF, XTEN, orspacer, each of the CF, XTEN, and spacer are the same or different. Insome embodiments, the CFXTEN is a monomeric fusion protein with a CFlinked to one XTEN polypeptide. In other embodiments, the CFXTEN is amonomeric fusion protein with a CF linked to two or more XTENpolypeptides. In still other embodiments, the CFXTEN is a monomericfusion protein with two or more CF linked to one XTEN polypeptide. Instill other embodiments, the CFXTEN is a monomeric fusion protein withtwo or more CF linked to two or more XTEN polypeptide. In still otherembodiment, the CFXTEN is a monomeric fusion protein with a single CF inwhich XTEN is located within the CF sequence (e.g., within a FIXsequence such as between one or more domains as illustrated in FIGS. 2and 5). Table 6 provides non-limiting examples of configurations thatare encompassed by the CFXTEN fusion proteins of the invention; numerousother variations will be apparent to the ordinarily skilled artisan,including the incorporation the spacer and cleavage sequences disclosedherein or known in the art.

TABLE 6 CFXTEN configurations Components* Configuration**Single CF; Single XTEN CF-XTEN XTEN-CF Single CF; Multiple XTENXTEN-CF-XTEN CF-XTEN-XTEN XTEN-XTEN-CF XTEN-CF-XTEN-XTENXTEN-XTEN-CF-XTEN XTEN-XTEN-CF-XTEN Multiple CF, Single XTEN CF-XTEN-CFXTEN-CF-CF CF-CF-XTEN CF-XTEN-CF-CF Multiple CF; Multiple XTENCF-XTEN-CF-XTEN XTEN-CF-XTEN-CF XTEN-XTEN-CF-XTEN-CF XTEN-XTEN-CF-CFCF-XTEN-XTEN-CF CF-CF-XTEN-XTEN CF-CF-XTEN-XTEN-CF CF-XTEN-CF-XTEN-CF*Characterized as single for 1 component or multiple for 2 or more ofthat component **Reflects N- to C-terminus configuration of the growthfactor and XTEN components

The invention contemplates CFXTEN fusion proteins compositionscomprising, but not limited to single or multiple CF selected from Table1 or Table 2 (or fragments or sequence variants thereof), single ormultiple XTEN selected from Table 4 (or sequence variants thereof) thatare in a configuration shown in Table 6. Non-limiting examples ofsequences of fusion proteins containing a single CF linked to a singleXTEN are presented in Table 41. In one embodiment, a CFXTEN compositionwould comprise a fusion protein having at least about 80% sequenceidentity compared to a CFXTEN from Table 41, alternatively at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity ascompared to a CFXTEN from Table 41. Generally, the resulting CFXTENretains at least a portion of the biological activity of thecorresponding CF not linked to the XTEN. In the foregoing fusionproteins hereinabove described in this paragraph, the CFXTEN fusionprotein can further comprise a cleavage sequence from Table 7; thecleavage sequence being located between the CF and the XTEN or betweenadjacent CF (if more than one CF is included in the CFXTEN). In somecases, the CFXTEN comprising the cleavage sequences will also have oneor more spacer sequence amino acids between the CF and the cleavagesequence or the XTEN and the cleavage sequence to facilitate access ofthe protease; the spacer amino acids comprising any natural amino acid,including glycine and alanine as preferred amino acids. Non-limitingexamples of CFXTEN comprising CF, XTEN, cleavage sequence(s) and spaceramino acids are presented in Table 42. However, the invention alsocontemplates substitution of any of the CF sequences of Tables 1 and 2for a CF sequence of Table 42, substitution of any XTEN sequence ofTable 4 for an XTEN sequence of Table 42, and substitution of anycleavage sequence of Table 7 for a cleavage sequence of Table 42. InCFXTEN embodiments having one or more cleavage sequences, the CFcomponent either becomes biologically active or has an increase inactivity upon its release from the XTEN by cleavage of the cleavagesequence(s), described more fully below.

In one embodiment of the CFXTEN composition, the invention provides afusion protein of formula I:

(XTEN)_(x)-CF-(XTEN)_(y)  I

wherein independently for each occurrence, CF is a coagulation factor; xis either 0 or 1 and y is either 0 or 1 wherein x+y≥1; and XTEN is anextended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesa fusion protein of formula II:

(XTEN)_(x)-(CF)-(S)_(y)-(XTEN)_(y)  II

wherein independently for each occurrence, CF is a coagulation factor a;S is a spacer sequence having between 1 to about 50 amino acid residuesthat can optionally include a cleavage sequence; x is either 0 or 1 andy is either 0 or 1 wherein x+y≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein, wherein the fusion protein is of formulaIII:

(XTEN)_(x)-(S)_(x)-(CF)-(S)_(y)-(XTEN)_(y)  III

wherein independently for each occurrence, CF is a coagulation factor; Sis a spacer sequence having between 1 to about 50 amino acid residuesthat can optionally include a cleavage sequence; x is either 0 or 1 andy is either 0 or 1 wherein x+y≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula IV:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(XTEN)_(w)-(AP)-(XTEN)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  IV

wherein independently for each occurrence, Gla is a Gla domain of FIX;EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP is anactivator peptide of FIX; PRO is a protease domain of FIX; S is a spacersequence having between 1 to about 50 amino acid residues that canoptionally include a cleavage sequence; u is either 0 or 1; v is either0 or 1; x is either 0 or 1; y is either 0 or 1, z is either 0 or 1, withthe proviso that u+v+x+z≥1; and XTEN is an extended recombinantpolypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula V:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(AP1)-(XTEN)_(w)-(AP2)-(XTEN)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  V

wherein independently for each occurrence, Gla is a Gla domain of FIX;EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP1 is theN-terminal sequence portion of the activator peptide domain of FIX thatincludes a first native cleavage sequence of the AP domain; AP2 is theC-terminal sequence portion of the activator peptide domain of FIX thatincludes a second native cleavage sequence of the AP domain; PRO is aprotease domain of FIX; S is a spacer sequence having between 1 to about50 amino acid residues that can optionally include a cleavage sequence;u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1;y is either 0 or 1; z is either 0 or 1 with the proviso thatu+v+w+x+z≥1; and XTEN is an extended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula VI:

(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(XTEN)_(w)-(Pro)-(S)_(x)-(XTEN)_(y)  VI

wherein independently for each occurrence, Gla is a Gla domain of FVII;EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is aprotease domain of FVII; S is a spacer sequence having between 1 toabout 50 amino acid residues that can optionally include a cleavagesequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; yis either 0 or 1; and XTEN is an extended recombinant polypeptide.

In another embodiment of the CFXTEN composition, the invention providesan isolated fusion protein of formula VII:

(Gla)-(XTEN)_(t)-(EGF1)-(XTEN)_(u)-(EGF2)-(AP1)_(v)-(XTEN)_(w)-(AP2)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  VII

wherein independently for each occurrence, Gla is a Gla domain of FVII;EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is aprotease domain of FVII; AP1 is the N-terminal sequence portion of theactivator peptide domain of FIX that includes the native cleavagesequence; AP2 is the C-terminal sequence portion of the activatorpeptide domain of FIX that includes the native cleavage sequence; S is aspacer sequence having between 1 to about 50 amino acid residues thatcan optionally include a cleavage sequence; t is either 0 or 1; u iseither 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or1; z is either 0 or 1; and XTEN is an extended recombinant polypeptide.In the embodiment, the factor VII variant includes can include one orboth cleavage sequences from the activator peptide domain of factor IX;e.g., a sequence of at least about 2, or at least about 3, or at leastabout 4, or at least about 5 amino acids that flank the R145-A146cleavage site (e.g., the sequence TSKLTRAETVFP (SEQ ID NO: 3) in thecase of 5 flanking amino acids) and the sequence of at least about 2, orat least about 3, or at least about 4, or at least about 5 amino acidsthat flank the R180-V181 cleavage site (e.g., the sequence FNDFTRVVGGED(SEQ ID NO: 85) in the case of 5 flanking amino acids, as described morefully above. The invention also contemplates substitution of any of theother cleavage sequences of Table 7 for the AP sequences of the factorVII variant.

The embodiments of formulae V and VI encompass CFXTEN configurations offactor IX and factor VII, respectively, wherein one or more XTEN oflengths ranging from about 36 amino acids to ≥1000 amino acids (e.g.,sequences selected from Tables 4, and 9-13) are inserted and linkedbetween adjoining domains of the factor IX or the factor VII sequence,respectively. The invention contemplates all possible permutations ofinsertions of XTEN between the domains of either FIX or FVII withoptional linking of an additional XTEN to the C-terminus of the FIX orthe FVII, optionally via an additional cleavage sequence selected fromTable 7, resulting in a CFXTEN composition; non-limiting examples ofwhich are portrayed in FIGS. 2, 5 and 6. In the foregoing embodimentshereinabove described in this paragraph, the CFXTEN fusion proteins canbe evaluated for retention of biological activity (including aftercleavage of any incorporated XTEN-releasing cleavage sites) using anyappropriate in vitro assay disclosed herein (e.g., the assays of Table40 or the assays described in the Examples), to determine thesuitability of the configuration for use as a therapeutic agent in thetreatment of a coagulation-factor related disease, disorder orcondition.

In some embodiments, administration of a therapeutically effectiveamount of a fusion protein of one of formulae I-VII to a subject in needthereof results in an increase of at least two-fold in the terminalhalf-life, or at least three-fold, or at least four-fold, or at leastfive-fold, or at least 10-fold, or at least 20-fold, or at least40-fold, or at least 100-fold increase in the terminal half-life for thefusion protein compared to the corresponding CF not linked to the XTENand administered at a comparable amount administered to a subject. Insome embodiments, administration of a therapeutically effective amountof a fusion protein of one of formulae I-VII to a subject in needthereof results in a gain in time of at least two-fold, or at leastthree-fold, or at least four-fold, or at least five-fold, or at least10-fold, or at least 20-fold, or at least 40-fold, or at least 100-foldor more spent within a therapeutic window for the fusion proteincompared to the corresponding CF not linked to the XTEN and administeredat a comparable amount administered to a subject. In other embodiments,administration of a therapeutically effective dose of a fusion proteinof one of formulae I-VII to a subject in need thereof can result in again in time between consecutive doses necessary to maintain atherapeutically effective blood level of the fusion protein of at least48 h, or at least 72 h, or at least about 96 h, or at least about 120 h,or at least about 7 days, or at least about 14 days, or at least about21 days between consecutive doses compared to a CF not linked to XTENand administered at a comparable dose.

Any spacer sequence group optionally is introduced to a subject fusionprotein encompassed by the invention. The spacer is provided to enhanceexpression of the fusion protein from a host cell or to decrease sterichindrance such that the CF component may assume its desired tertiarystructure and/or interact appropriately with its target substrate. Forspacers and methods of identifying desirable spacers, see, for example,George, et al. (2003) Protein Engineering 15:871-879, specificallyincorporated by reference herein. In one embodiment, the spacercomprises one or more peptide sequences that are between 1-50 amino acidresidues in length, or about 1-25 residues, or about 1-10 residues inlength. Spacer sequences, exclusive of cleavage sites, can comprise anyof the 20 natural L amino acids, and will preferably comprisehydrophilic amino acids that are sterically unhindered that can include,but not be limited to, glycine (G), alanine (A), serine (S), threonine(T), glutamate (E) and proline (P). In some cases, the spacer can bepolyglycines or polyalanines, or is predominately a mixture ofcombinations of glycine and alanine residues. The spacer polypeptideexclusive of a cleavage sequence is largely to substantially devoid ofsecondary structure; e.g., less than about 10%, or less than about 5% asdetermined by the Chou-Fasman and/or GOR algorithms. In one embodiment,a spacer sequence in a CFXTEN fusion protein composition furthercontains one or more cleavage sequences, which are identical ordifferent, wherein the cleavage sequence may be acted on by a proteaseto release the CF from the fusion protein.

In some embodiments, the incorporation of the cleavage sequence into theCFXTEN is designed to permit release of a CF that becomes active or moreactive upon its release from the XTEN; e.g., the enzymatic activity ofthe CF component increases. In one embodiment of the foregoing, the CFthat becomes active after release is a FIX or a sequence variantthereof. In another embodiment of the foregoing, the CF that becomesactive after release is a FVII or a sequence variant thereof. Thecleavage sequences are located sufficiently close to the CF sequences,generally within 18, or within 12, or within 6, or within 2 amino acidsof the CF sequence terminus, such that any remaining residues attachedto the CF after cleavage do not appreciably interfere with the activity(e.g., such as binding to a ligand or substrate) of the CF, yet providesufficient access to the protease to be able to effect cleavage of thecleavage sequence. In some embodiments, the cleavage site is a sequencethat can be cleaved by a protease endogenous to the mammalian subjectsuch that the CFXTEN can be cleaved after administration to a subject.In such cases, the CFXTEN can serve as a prodrug or a circulating depotfor the CF. In one embodiment, the CF that is released from the fusionprotein by cleavage of the cleavage sequence exhibits at least about atwo-fold, or at least about a three-fold, or at least about a four-fold,or at least about a five-fold, or at least about a six-fold, or at leastabout a eight-fold, or at least about a ten-fold, or at least about a20-fold increase in enzymatic activity for its native substrate comparedto the intact CFXTEN fusion protein.

Examples of cleavage sites contemplated by the invention include, butare not limited to, a polypeptide sequence cleavable by a mammalianendogenous protease selected from FXIa, FXIIa, kallikrein, FVIIa, FIXa,FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 orMMP-20, or by non-mammalian proteases such as TEV, enterokinase,PreScission™ protease (rhinovirus 3C protease), and sortase A. Sequencesknown to be cleaved by the foregoing proteases and others are known inthe art. Exemplary cleavage sequences and cut sites within the sequencesare presented in Table 7, as well as sequence variants thereof. Forexample, thrombin (activated clotting factor II) acts on the sequenceLTPRSLLV (SEQ ID NO: 86) [Rawlings N. D., et al. (2008) Nucleic AcidsRes., 36: D320], which is cut after the arginine at position 4 in thesequence. Active FIIa is produced by cleavage of FII by FXa in thepresence of phospholipids and calcium and is down stream from factor IXin the coagulation pathway. Once activated, its natural role incoagulation is to cleave fibrinogen, which then in turn, begins clotformation. FIIa activity is tightly controlled and only occurs whencoagulation is necessary for proper hemostasis. However, as coagulationis an on-going process in mammals, by incorporation of the LTPRSLLV (SEQID NO: 86) sequence into the CFXTEN between and linking the CF and theXTEN components, the XTEN is removed from the adjoining CF concurrentwith activation of either the extrinsic or intrinsic coagulationpathways when coagulation is required physiologically, thereby releasingCF over time. Similarly, incorporation of other cleavage sequences intoCFXTEN that are acted upon by endogenous proteases, particularly thosesusceptible to the activated clotting proteins listed in Table 7, wouldprovide for sustained release of CF that, in certain embodiments of theCFXTEN, provide a higher degree of activity for the CF componentreleased from the intact form of the CFXTEN. In one embodiment, theinvention provides CFXTEN comprising one or more cleavage sequencesoperably positioned to release the CF from the fusion protein uponcleavage, wherein the one or more cleavage sequences has at least about86%, or at least about 92% or greater sequence identity to a sequenceselected from Table 7. In another embodiment, the CFXTEN comprising acleavage sequence would have at least about 80%, or at least about 85%,or at least about 90%, or at least about 95%, or at least about 96%, orat least about 97%, or at least about 98%, or at least about 99%sequence identity compared to a sequence selected from Table 42.

In some embodiments, only the two or three amino acids flanking bothsides of the cut site (four to six amino acids total) are incorporatedinto the cleavage sequence that, in turn, is incorporated into theCFXTEN of the embodiments. In other embodiments, the known cleavagesequence have one or more deletions or insertions or one or two or threeamino acid substitutions for any one or two or three amino acids in theknown sequence, wherein the deletions, insertions or substitutionsresult in reduced or enhanced susceptibility but not an absence ofsusceptibility to the protease, resulting in an ability to tailor therate of release of the CF from the XTEN. Exemplary substitutions areshown in Table 7.

TABLE 7 Protease Cleavage Sequences Exemplary Protease Acting CleavageSEQ ID SEQ ID Upon Sequence Sequence NO: Minimal Cut Site* NO: FXIaKLTR↓AET 87 KD/FL/T/R↓VA/VE/GT/GV FXIa DFTR↓VVG 88 KD/FL/T/R↓VA/VE/GT/GVFXIIa TMTR↓IVGG 89 NA Kallikrein SPFR↓STGG 90 -/-/FL/RY↓SR/RT/-/- FVIIaLQVR↓IVGG 91 NA FIXa PLGR↓IVGG 92 -/-/G/R↓-/-/-/- FXa IEGR↓TVGG 93IA/E/GFP/R↓STI/VFS/-/G FIIa (thrombin) LTPR↓SLLV 94 -/-/PLA/R↓SAG/-/-/-Elastase-2 LGPV↓SGVP 95 -/-/-/VIAT↓-/-/-/- Granzyme-B VAGD↓SLEE 96V/-/-/D↓-/-/-/- MMP-12 GPAG↓LGGA 97 G/PA/-/G↓L/-/G/- 98 MMP-13 GPAG↓LRGA99 G/P/-/G↓L/-/GA/- 100 MMP-17 APLG↓LRLR 101 -/PS/-/-↓LQ/-/LT/- MMP-20PALP↓LVAQ 102 NA TEV ENLYFQ↓G 103 ENLYFQ↓G/S 104 Enterokinase DDDK↓IVGG105 DDDK↓IVGG 106 Protease 3C LEVLFQ↓GP 107 LEVLFQ↓GP 108(PreScission ™) Sortase A LPKT↓GSES 109 L/P/KEAD/T↓G/-/EKS/S 110↓indicates cleavage site NA: not applicable *the listing of multipleamino acids before, between, or after a slash indicate alternative aminoacids that can be substituted at the position; “-” indicates that anyamino acid may be substituted for the corresponding amino acid indicatedin the middle column

(a) Pharmacokinetic Properties of CFXTEN

The invention provides CFXTEN fusion proteins with enhancedpharmacokinetics compared to the CF not linked to XTEN. Thepharmacokinetic properties of a CF that can be enhanced by linking agiven XTEN to the CF include, but are not limited to, terminalhalf-life, area under the curve (AUC), C_(max), volume of distribution,and bioavailability; properties that provide enhanced utility in thetreatment of coagulation factor-related disorders, diseases and relatedconditions. As a result of the enhanced properties, the CFXTEN, whenused at the dose and dose regimen determined to be appropriate for thecomposition by the methods described herein, can achieve a circulatingconcentration resulting in a desired pharmacologic effect, yet staywithin the safety range for biologically active component of thecomposition for an extended period of time compared to a comparable doseof the CF not linked to XTEN. In such cases, the CFXTEN remains withinthe therapeutic window for the fusion protein composition for theextended period of time compared to a CF not liked to XTEN andadministered to a subject at a comparable dose. As used herein, a“comparable dose” means a dose with an equivalent moles/kg for theactive CF pharmacophore (e.g., FIX or FVII) that is administered to asubject in a comparable fashion. It will be understood in the art that a“comparable dosage” of CFXTEN fusion protein would represent a greaterweight of agent but would have essentially the same mole-equivalents ofCF in the dose of the fusion protein administered.

In some embodiments, the CFXTEN with enhanced pharmacokinetic propertiescan be a sequence that has at least about 80% sequence identity, oralternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identitycompared to a protein sequence selected from any one of Tables 41, 42,or 43. In other embodiments, the CFXTEN with enhanced pharmacokineticproperties can comprise a CF sequence that has at least about 80%sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%sequence identity compared to a sequence from Table 1 or from Table 2,linked to one or more XTEN that has at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequenceidentity compared to a sequence from Table 4. For the inventivecompositions, CFXTEN with a longer terminal half-life are generallypreferred, so as to improve patient convenience, to increase theinterval between doses and to reduce the amount of drug required toachieve a sustained effect. In the embodiments hereinabove described inthis paragraph the administration of the fusion protein results in animprovement in at least one of the parameters (disclosed herein as beinguseful for assessing the subject diseases, conditions or disorders)using a lower unit dose in moles of fusion protein compared to thecorresponding CF component not linked to the fusion protein andadministered at a comparable unit dose or dose regimen to a subject. Inthe foregoing embodiments, the total dose in moles administered toachieve the improvement is at least about three-fold lower, or at leastabout four-fold, or at least about five-fold, or at least aboutsix-fold, or at least about eight-fold, or at least about 10-fold lowercompared to the corresponding CF component not linked to the fusionprotein.

As described more fully in the Examples pertaining to pharmacokineticcharacteristics of fusion proteins comprising XTEN, it was observed thatincreasing the length of the XTEN sequence confers a disproportionateincrease in the terminal half-life of a fusion protein comprising theXTEN. Accordingly, the invention provides CFXTEN fusion proteinscomprising XTEN wherein the XTEN is selected to provide a targetedhalf-life for the CFXTEN composition administered to a subject. In someembodiments, the invention provides monomeric fusion proteins comprisingXTEN wherein the XTEN is selected to confer an increase in the terminalhalf-life for the CFXTEN administered to a subject, compared to thecorresponding CF not linked to the fusion protein and administered at acomparable dose, wherein the increase is at least about two-fold longer,or at least about three-fold, or at least about four-fold, or at leastabout five-fold, or at least about six-fold, or at least aboutseven-fold, or at least about eight-fold, or at least about nine-fold,or at least about ten-fold, or at least about 15-fold, or at least a20-fold, or at least a 40-fold, or at least a 80-fold, or at least a100-fold or greater an increase in terminal half-life compared to the CFnot linked to the fusion protein. Exogenously administered factor IX hasbeen reported to have a terminal half-life in humans of approximately18-24 hours (Morfini, M. Blood Transfus. (2008) 6(s2): s21-s25) andexogenously administered factor VII is reported to have a terminalhalf-life of approximately 4-6 hours (Klitgaard T, Br J Clin Pharmacol(2008) 65(1):3-11), whereas various CFXTEN compositions disclosed hereinthat have been experimentally administered to animals, as described inthe Examples, have resulted in terminal half-life values considerablylonger. In open embodiment, the present invention provides CFXTEN fusionproteins that exhibits an increase in ACU of at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90%, or at least about a 100%, or at least about 150%, or atleast about 200%, or at least about 300%, or at least about 500%, or atleast about 1000%, or at least about a 2000% compared to thecorresponding CF not linked to the XTEN and administered to a subject ata comparable dose. The pharmacokinetic parameters of a CFXTEN can bedetermined by standard methods involving dosing, the taking of bloodsamples at times intervals, and the assaying of the protein using ELISA,HPLC, radioassay, or other methods known in the art or as describedherein, followed by standard calculations of the data to derive thehalf-life and other PK parameters.

The enhanced PK parameters allow for reduced dosing of the CFXTENcompositions, compared to CF not linked to XTEN. In some embodiments, asmaller molar amount of about two-fold less, or about three-fold less,or about four-fold less, or about five-fold less, or about six-foldless, or about eight-fold less, or about 10-fold less or greater of thefusion protein is administered in comparison to the corresponding CF notlinked to the XTEN under a dose regimen needed to maintain hemostasis,and the fusion protein achieves a comparable area under the curve as thecorresponding molar amount of the CF not linked to the XTEN. In otherembodiments, the fusion protein has a less frequent administrationregimen of about every two days, about every seven days, about every 14days, about every 21 days, or about monthly of the fusion proteinadministered to a subject, compared to the daily administration of anotherwise same dose amount of the corresponding CF not linked to theXTEN, and the fusion protein achieves a comparable area under the curveas the corresponding CF not linked to the XTEN. In yet otherembodiments, an accumulative smaller molar amount of about 5%, or about10%, or about 20%, or about 40%, or about 50%, or about 60%, or about70%, or about 80%, or about 90% less of the fusion protein isadministered to a subject in comparison to the corresponding molaramount of the CF not linked to the XTEN under a dose regimen needed tomaintain hemostasis, yet the fusion protein achieves at least acomparable area under the curve as the corresponding CF not linked tothe XTEN. The accumulative smaller molar amount is measure for a periodof at least about one week, or about 14 days, or about 21 days, or aboutone month.

The invention further provides CFXTEN comprising a CF molecule separatedfrom the XTEN sequence by one or more cleavage sequences; e.g., asequence from Table 7. In some embodiments, the intact CFXTENcomposition has less activity but a longer half-life in its intact formcompared to a corresponding CF not linked to the XTEN, but is designedsuch that upon administration to a subject, the CF component isgradually released from the fusion protein by cleavage at the cleavagesequence(s) by endogenous proteases, whereupon the CF component exhibitsactivity, i.e., the ability to effectively bind to and activate itstarget coagulation protein substrate. In non-limiting examples, theCFXTEN with a cleavage sequence has about 80% sequence identity comparedto a sequence from Table 42, or about 85%, or about 90%, or about 95%,or about 97%, or about 98%, or about 99% sequence identity compared to asequence from Table 42. Accordingly, the CFXTEN of the foregoingembodiments in this paragraph serve as prodrugs or a circulating depot,resulting in a longer terminal half-life compared to CF not linked tothe XTEN. In such cases, a higher concentration of CFXTEN can beadministered to a subject, compared to the corresponding CF not linkedto XTEN because a smaller proportion of the circulating composition isactive.

(b) Pharmacology and Pharmaceutical Properties of CFXTEN

The present invention provides CFXTEN compositions comprising CFcovalently linked to XTEN that can have enhanced properties compared toCF not linked to XTEN, as well as methods to enhance the therapeuticand/or biologic activity or effect of the respective two CF componentsof the compositions. In addition, the invention provides CFXTENcompositions with enhanced properties compared to those art-known fusionproteins containing albumin, immunoglobulin polypeptide partners,polypeptides of shorter length and/or polypeptide partners withrepetitive sequences. In addition, CFXTEN fusion proteins providesignificant advantages over chemical conjugates, such as pegylatedconstructs, notably the fact that recombinant CFXTEN fusion proteins canbe made in bacterial cell expression systems, which can reduce time andcost at both the research and development and manufacturing stages of aproduct, as well as result in a more homogeneous, defined product withless toxicity for both the product and metabolites of the CFXTENcompared to pegylated conjugates.

As therapeutic agents, the CFXTEN possesses a number of advantages overtherapeutics not comprising XTEN, including one or more of the followingnon-limiting exemplary enhanced properties: increased solubility,increased thermal stability, reduced immunogenicity, increased apparentmolecular weight, reduced renal clearance, reduced proteolysis, reducedmetabolism, enhanced therapeutic efficiency, a lower effectivetherapeutic dose, increased bioavailability, increased time betweendosages capable of maintaining blood levels within the therapeuticwindow for the CF, a “tailored” rate of absorption when administeredsubcutaneously or intramuscularly, enhanced lyophilization stability,enhanced serum/plasma stability, increased terminal half-life, increasedsolubility in blood stream, decreased binding by neutralizingantibodies, decreased active clearance, reduced side effects, retentionof substrate binding affinity, stability to degradation, stability tofreeze-thaw, stability to proteases, stability to ubiquitination, easeof administration, compatibility with other pharmaceutical excipients orcarriers, persistence in the subject, increased stability in storage(e.g., increased shelf-life), reduced toxicity in an organism orenvironment and the like. The net effect of the enhanced properties isthat the use of a CFXTEN composition can result in enhanced therapeuticand/or biologic effect compared to a CF not linked to XTEN or result inimproved patient compliance when administered to a subject with acoagulation factor-related disease or disorder.

Specific assays and methods for measuring the physical and structuralproperties of expressed proteins are known in the art, including methodsfor determining properties such as protein aggregation, solubility,secondary and tertiary structure, melting properties, contamination andwater content, etc. Such methods include analytical centrifugation, EPR,HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, lightscattering, capillary electrophoresis, circular dichroism, differentialscanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-sizeexclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visiblespectroscopy; several of which are applied to the inventive CFXTEN asdescribed in the Examples. Additional methods are disclosed in Arnau etal, Prot Expr and Purif (2006) 48, 1-13. Application of these methods toelucidate the enhanced properties of the compositions of the inventionis within the grasp of a person skilled in the art.

In one embodiment, XTEN as a fusion partner increases the solubility ofthe CF payload. Accordingly, where enhancement of the pharmaceutical orphysicochemical properties of the CF is desirable, such as the degree ofaqueous solubility or stability, the length and/or the motif familycomposition of the XTEN sequences incorporated into the fusion proteinmay each be selected to confer a different degree of solubility and/orstability on the respective fusion proteins such that the overallpharmaceutical properties of the CFXTEN composition are enhanced. TheCFXTEN fusion proteins can be constructed and assayed, using methodsdescribed herein, to confirm the physicochemical properties and the XTENadjusted, as needed, to result in the desired properties. In oneembodiment, the XTEN sequence of the CFXTEN is selected such that thefusion protein has an aqueous solubility that is within at least about25% greater compared to a CF not linked to the fusion protein, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 75%, or at least about 100%, or at least about 200%, or atleast about 300%, or at least about 400%, or at least about 500%, or atleast about 1000% greater than the corresponding CF not linked to thefusion protein.

The invention provides methods to produce and recover expressed CFXTENfrom a host cell with enhanced solubility and ease of recovery comparedto CF not linked to XTEN. In some embodiments, the method includes thesteps of transforming a eukaryotic host cell with a polynucleotideencoding a CFXTEN with one or more XTEN components of cumulativesequence length greater than about 200, or greater than about 400, orgreater than about 600, or greater than about 800 amino acid residues,expressing the CFXTEN fusion protein in the host cell, and recoveringthe expressed fusion protein in soluble form. In the embodimentshereinabove described in this paragraph, the XTEN of the CFXTEN fusionproteins can have at least about 80% sequence identity, or about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99%, to about 100%sequence identity compared to one or more XTEN selected from Table 4 andthe CF can have at least about 80% sequence identity, or about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99%, or 100% sequenceidentity compared to a CF selected from Table 1 or Table 2 and theCFXTEN components can be in an N- to C-terminus configuration selectedfrom formulas I-VII.

In one embodiment, the invention provides CFXTEN compositions andmethods to produce the compositions that can maintain the CF componentwithin a therapeutic window for a greater period of time compared tocomparable dosages of the corresponding CF not linked to XTEN. It willbe understood in the art that a “comparable dosage” of CFXTEN fusionprotein would represent a greater weight of agent but would have thesame approximate mole-equivalents of CF in the dose of the fusionprotein and/or would have the same approximate molar concentrationrelative to the CF. The method to produce the compositions that canmaintain the CF component within a therapeutic window includes the stepsof selecting the XTEN appropriate for conjugation to a CF to provide thedesired pharmacokinetic properties in view of a given dose and doseregimen, administration of the CFXTEN to subjects in need thereof,followed by assays to verify the pharmacokinetic properties, theactivity of the CFXTEN fusion protein, and the safety of theadministered composition. By the methods, CFXTEN provided herein allowfor increased efficacy of the administered composition by maintainingthe circulating concentrations of the CF within the therapeutic windowfor an enhanced period of time. As used herein, “therapeutic window”means that the amount of drug or biologic as a blood or plasmaconcentration range, which provides efficacy or a desired pharmacologiceffect over time for the disease or condition without unacceptabletoxicity, i.e., the range of the circulating blood concentrationsbetween the minimal amount to achieve any positive therapeutic effectand the maximum amount which results in a response that is the responseimmediately before toxicity to the subject (at a higher dose orconcentration). Additionally, therapeutic window generally encompassesan aspect of time; the maximum and minimum concentration that results ina desired pharmacologic effect over time that does not result inunacceptable toxicity or adverse events. A dosed composition that stayswithin the therapeutic window for the subject could also be said to bewithin the “safety range.”

The characteristics of CFXTEN compositions of the invention, includingfunctional characteristics or biologic and pharmacologic activity andparameters that result, can be determined by any suitable screeningassay known in the art for measuring the desired characteristic. Theinvention provides methods to assay the CFXTEN fusion proteins ofdiffering composition or configuration in order to provide CFXTEN withthe desired degree of biologic and/or therapeutic activity, as well assafety profile. Specific in vivo and ex vivo biological assays are usedto assess the activity of each configured CFXTEN and/or CF component tobe incorporated into CFXTEN, including but not limited to the assays ofthe Examples, those assays of Table 40, as well as the following assaysor other such assays known in the art for assaying the properties andeffects of CF. Functional assays can be conducted that allowdetermination of coagulation activity, such as prothrombin (PT) andactivated partial prothrombin (aPTT) assays (Belaaouaj A A et al., J.Biol. Chem. (2000) 275:27123-8; Diaz-Collier J A. Haemost (1994)71:339-46), blood clotting time (WBCT), thrombelastography, or bleedingtime assays. Other possible assays may determine the binding affinity ofa CFXTEN for the target substrate of the corresponding CF can be assayedusing binding or competitive binding assays, such as Biacore assays withchip-bound receptors or binding proteins or ELISA assays, as describedin U.S. Pat. No. 5,534,617, assays described in the Examples herein,radio-receptor assays, or other assays known in the art. The foregoingassays can also be used to assess CF sequence variants (assayed assingle components or as CFXTEN fusion proteins) and can be compared tothe native CF to determine whether they have the same degree of biologicactivity as the native CF, or some fraction thereof such that they aresuitable for inclusion in CFXTEN; e.g., at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% of the activitycompared to the native CF.

Dose optimization is important for all drugs, especially for those witha narrow therapeutic window. For example, a standardized single dose ofCF for all patients presenting with a diverse symptoms or abnormalclinical parameters may not always be effective. A consideration ofthese factors is well within the purview of the ordinarily skilledclinician for the purpose of determining the therapeutically orpharmacologically effective amount of the CFXTEN, versus that amountthat would result in unacceptable toxicity and place it outside of thesafety range, or insufficient potency such that clinical improvement isnot achieved.

In many cases, the therapeutic window for CF in subjects of differentages or degree of disease have been established and are available inpublished literature or are stated on the drug label for approvedproducts containing the CF. In other cases, the therapeutic window canbe established for new compositions, including those CFXTEN of thedisclosure. The methods for establishing the therapeutic window for agiven composition are known to those of skill in the art (see, e.g.,Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11^(th)Edition, McGraw-Hill (2005)). For example, by using dose-escalationstudies in subjects with the target disease or disorder to determineefficacy or a desirable pharmacologic effect, appearance of adverseevents, and determination of circulating blood levels, the therapeuticwindow for a given subject or population of subjects can be determinedfor a given drug or biologic, or combinations of biologics or drugs. Thedose escalation studies can evaluate the activity of a CFXTEN throughmetabolic studies in a subject or group of subjects that monitorphysiological or biochemical parameters, as known in the art or asdescribed herein for one or more parameters associated with themetabolic disease or disorder, or clinical parameters associated with abeneficial outcome for the particular indication, together withobservations and/or measured parameters to determine the no effect dose,adverse events, maximum tolerated dose and the like, together withmeasurement of pharmacokinetic parameters that establish the determinedor derived circulating blood levels. The results can then be correlatedwith the dose administered and the blood concentrations of thetherapeutic that are coincident with the foregoing determined parametersor effect levels. By these methods, a range of doses and bloodconcentrations can be correlated to the minimum effective dose as wellas the maximum dose and blood concentration at which a desired effectoccurs and above which toxicity occurs, thereby establishing thetherapeutic window for the dosed therapeutic. Blood concentrations ofthe fusion protein (or as measured by the CF component) above themaximum is considered outside the therapeutic window or safety range.Thus, by the foregoing methods, a C_(min) blood level is established,below which the CFXTEN fusion protein would not have the desiredpharmacologic effect, and a C_(max) blood level is established thatwould represent the highest circulating concentration before reaching aconcentration that would elicit unacceptable side effects, toxicity oradverse events, placing it outside the safety range for the CFXTEN. Withsuch concentrations established, the frequency of dosing and the dosagecan be further refined by measurement of the C_(max) and C_(min) toprovide the appropriate dose and dose frequency to keep the fusionprotein(s) within the therapeutic window.

One of skill in the art can, by the means disclosed herein or by othermethods known in the art, confirm that the administered CFXTEN remainsin the therapeutic window for the desired interval or requiresadjustment in dose or length or sequence of XTEN. Further, thedetermination of the appropriate dose and dose frequency to keep theCFXTEN within the therapeutic window establishes the therapeuticallyeffective dose regimen; the schedule for administration of multipleconsecutive doses using a therapeutically effective dose of the fusionprotein to a subject in need thereof resulting in consecutive C_(max)peaks and/or C_(min) troughs that remain within the therapeutic windowand results in an improvement in at least one measured parameterrelevant for the target disease, disorder or condition. In some cases,the CFXTEN administered at an appropriate dose to a subject results inblood concentrations of the CFXTEN fusion protein that remains withinthe therapeutic window for a period at least about two-fold longercompared to the corresponding CF not linked to XTEN and administered ata comparable dose; alternatively at least about three-fold longer;alternatively at least about four-fold longer; alternatively at leastabout five-fold longer; alternatively at least about six-fold longer;alternatively at least about seven-fold longer; alternatively at leastabout eight-fold longer; alternatively at least about nine-fold longeror at least about ten-fold longer or greater compared to thecorresponding CF not linked to XTEN and administered at a comparabledose. As used herein, an “appropriate dose” means a dose of a drug orbiologic that, when administered to a subject, would result in adesirable therapeutic or pharmacologic effect and/or a bloodconcentration within the therapeutic window.

In one embodiment, the CFXTEN administered at a therapeuticallyeffective dose regimen results in a gain in time of at least aboutthree-fold longer; alternatively at least about four-fold longer;alternatively at least about five-fold longer; alternatively at leastabout six-fold longer; alternatively at least about seven-fold longer;alternatively at least about eight-fold longer; alternatively at leastabout nine-fold longer or at least about ten-fold longer between atleast two consecutive C_(max) peaks and/or C_(min) troughs for bloodlevels of the fusion protein compared to the corresponding biologicallyactive protein of the fusion protein not linked to the fusion proteinand administered at a comparable dose regimen to a subject. In anotherembodiment, the CFXTEN administered at a therapeutically effective doseregimen results in a comparable improvement in one, or two, or three ormore measured parameter using less frequent dosing or a lower totaldosage in moles of the fusion protein of the pharmaceutical compositioncompared to the corresponding biologically active protein component(s)not linked to the fusion protein and administered to a subject using atherapeutically effective dose regimen for the CF. The measuredparameters include any of the clinical, biochemical, or physiologicalparameters disclosed herein, or others known in the art for assessingsubjects with coagulation factor-related disorders.

In some embodiments, the CFXTEN fusion proteins of the invention retainat least about 0.05%, or about 0.1%, or about 1%, or about 10%, or about20%, or about 30%, or about 40%, or about 50%, or about 60%, or about70%, or about 80%, or about 90%, or about 95%, or about 98%, or about99% percent of the biological activity of the corresponding CF notlinked to the fusion protein with regard to an in vitro biologicactivity or pharmacologic effect known or associated with the use of thenative CF in the treatment and prevention of coagulation factor-relateddiseases, disorders, and conditions. Non-limiting examples of parametersor physiologic effects that can be assayed to assess the retainedactivity of the CFXTEN fusion proteins include prothrombin time (PT),activated partial thromboplastin time (aPTT), bleeding time, whole bloodclotting time (WBCT), and thrombelastography. In some embodiments, theactivity of the CF component is manifested by the intact CFXTEN fusionprotein, while in other cases the activity of the CF component isprimarily manifested upon cleavage and release of the CF from the fusionprotein by action of a protease that acts on a cleavage sequenceincorporated into the CFXTEN fusion protein, embodiments of which aredisclosed above. In the foregoing, the CFXTEN is designed to reduce thebinding affinity of the CF component for the coagulation substrate whenlinked to the XTEN but have restored or increased affinity when releasedfrom XTEN through the cleavage of cleavage sequence(s) incorporated intothe CFXTEN sequence, as described more fully above. In one embodiment ofthe foregoing, the invention provides an isolated fusion proteincomprising a FIX linked to XTEN by a cleavage sequence, wherein thefusion protein is substantially inactive prior to cleavage and whereinthe FIX released from the fusion protein by proteolytic cleavage at thecleavage sequence has biological activity that is at least about 60%, orat least about 70%, or at least about 80%, or at least about 90%, or atleast about 95% as active compared to native FIX not linked to XTEN.

In other cases, the CFXTEN can be designed to reduce active clearance ofthe CFXTEN to increase the terminal half-life of CFXTEN administered toa subject, while still retaining biological activity. The clearancemechanisms to remove CF from the circulation have yet to be fullyelucidated. Uptake, elimination, and inactivation of CFs can occur inthe circulatory system as well as in the extravascular space.Coagulation factors are complex proteins that interact with a largenumber of other proteins, lipids, and receptors, and many of theseinteractions can contribute to the elimination of CFs from thecirculation. For example, clearance mechanisms for FVII, aheterogeneously glycosylated protein, may include clearance by theliver. The effects of the gamma-carboxy glutamic acid (Gla) domain andthe sialic acid content of the protein on FVIIa clearance have beeninvestigated using a perfused liver model, with results suggesting thatcarbohydrate receptors (e.g. the asialoglycoprotein receptor, ASGPR) mayplay a role in FVIIa clearance. (Appa, R. S., et al. Thromb Haemost.(2010, epub May 27) 104(2)). In addition, CF can be lost throughextravasation and rapid active clearance, which is reflected in thegenerally poor bioavailability of intravenously administered coagulationfactors such as factor VIIa (see NovoSeven package insert). It isbelieved that the CFXTEN of the present invention has comparativelyhigher bioavailability achieved by reduced active clearance and/or byreduced extravasation by increasing the hydrodynamic radius, or apparentsize, of the molecule by the addition of unstructured XTEN to thecoagulation factor. In one embodiment, the invention provides CFXTENthat reduce clearance of the fusion protein by linking one or more XTENto the CF component of the fusion protein, wherein the fusion proteinhas an increase in apparent molecular weight factor of at least aboutfour-fold, or at least about five-fold, or at least about six-fold, orat least about seven-fold, or at least about eight-fold, or at leastabout ten-fold, or at least about twelve-fold, or at least aboutfifteen-fold, and wherein the terminal half-life of the CFXTEN whenadministered to a subject is increased at least about two-fold, or atleast about four-fold, or at least about eight-fold, or at least about10-fold, or at least about 20-fold, or at least about 30-fold, or atleast about 40-fold, or at least about 50-fold, or at least about60-fold, or at least about 70-fold, or at least about 80-fold or morecompared to the corresponding CF not linked to XTEN. In the foregoingembodiment, wherein at least two XTEN molecules are incorporated intothe CFXTEN, the XTEN can be identical or they can be of a differentsequence composition (and net charge) or length. Non-limiting examplesof the foregoing embodiment with two XTEN linked to a single FVII areillustrated in FIG. 6, and include the constructs (expressed using thedomains of FVII) Gla-EGF1-EGF2-AE144-Protease-AE864 orGla-EGF1-AE288-EGF2-Protease-AE864 (wherein the AE XTEN components haveapproximately a 17% net charge due to incorporated glutamic acid),Gla-EGF1-EGF2-AG144-Protease-AG864 or Gla-EGF1-AG144-EGF2-Protease-AE864(wherein the AG XTEN components have approximately no net charge). Notto be bound by a particular theory, the XTEN of the CFXTEN compositionswith the higher net charge are expected, as described above, to haveless non-specific interactions with various negatively-charged surfacessuch as blood vessels, tissues, or various receptors, which wouldfurther contribute to reduced active clearance. Conversely, the XTEN ofthe CFXTEN compositions with a low (or no) net charge are expected tohave a higher degree of interaction with surfaces that, whilecontributing to active clearance, can potentiate the activity of theassociated coagulation factor, given the known contribution of cell(e.g., platelets) and vascular surfaces to the coagulation process andthe intensity of activation of coagulation factors (Zhou, R., et al.,Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry(2000) 39(32):9850-9858). Thus, the invention provides CFXTEN in whichthe degree of potency, bioavailability, and half-life can be tailored bythe selection and placement of the type and length of the XTEN in theCFXTEN compositions. Accordingly, the invention contemplatescompositions in which a CF from Table 1 or from Table 2 and XTEN fromTable 4 are substituted for the respective components of the foregoingexamples, and are produced, for example, in a configuration from Table 6or from formulas I-VII such that the construct has reduced clearancecompared to an alternative configuration of the respective components.In some embodiments, the foregoing method for increasing the terminalhalf-life provides configured CFXTEN that can result in an increase inthe terminal half-life of at least about 30%, or about 50%, or about75%, or about 100%, or about 150%, or about 200%, or about 300%, orabout 400% or more compared to the half-life of a CFXTEN in a secondconfiguration where active clearance is not reduced. The inventionfurther takes advantage of the fact that certain ligands wherein reducedbinding to a clearance receptor, either as a result of a decreasedon-rate or an increased off-rate, may be effected by the obstruction ofeither the N- or C-terminus and using that terminus as the linkage toanother polypeptide of the composition, whether another molecule of aCF, an XTEN, or a spacer sequence results in the reduced binding. Thechoice of the particular configuration of the CFXTEN fusion proteinreduces the degree of binding to a clearance receptor such that areduced rate of active clearance is achieved.

In cases where a reduction in active clearance is desired but retentionof at least a portion of the biological activity is also desired, theCFXTEN is designed to retain sufficient biologic activity for the intactmolecule. Thus, in one embodiment, the invention provides a CFXTENconfigured such that the biologic activity of the CFXTEN is in the rangeof about 0.01%-40%, or about 0.01%-30%, or about 0.01%-20%, or about0.01%40 of the biological activity compared to the corresponding nativecoagulation factor. The biological activity of the configured CFXTEN isthus reduced by at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, or at least about 95%, or at leastabout 99%, or at least about 99.99% as compared to the biologicalactivity of the corresponding native coagulation factor not linked toXTEN, determined under comparable conditions. In the foregoingembodiments, the biological activity of the configured CFXTEN for thetarget receptor is “substantially reduced” compared to a correspondingnative CF not linked to XTEN. Accordingly, the present inventionprovides compositions and methods to produce compositions with reducedbiological activity but increased half-life by configuring the CFXTEN,examples of which are provided above, so as to be able to provide adesired in vivo biological response yet avoid active clearancemechanisms. The increased half-life permits higher dosages and reducedfrequency of dosing compared to CF not linked to XTEN or compared toCFXTEN configurations wherein the fusion protein is subject tocoagulation factor clearance mechanisms.

VI). Uses of the Compositions of the Present Invention

In another aspect, the invention provides a method for achieving abeneficial effect in bleeding disorders and/or in a coagulationfactor-related disease, disorder or condition mediated by FIX or FVII.As used herein, “coagulation factor-related diseases, disorders orconditions” is intended to include, but is not limited to bleedingdisorders (e.g., defective platelet function, thrombocytopenia or vonWillebrand's disease), coagulopathies (any disorder of bloodcoagulation, including coagulation factor deficiencies), hemophilia B(aka Christmas disease), factor IX-related bleeding disorders, factorVII deficiency, hemophilia A, vascular injury, uncontrolled bleeding insubjects not suffering from hemophilia, bleeding from trauma or surgery,bleeding due to anticoagulant therapy, and bleeding due to liver diseaseor conditions that can be ameliorated or corrected by administration ofFIX or FVII to a subject. The present invention addresses disadvantagesand/or limitations of other methods of treatment using factor IX orfactor VII preparations that have a relatively short terminal half-lifeand/or a narrow therapeutic window.

In some embodiments, the invention provides methods for treating asubject, such as a human, with a coagulation factor-related disease,disorder or condition comprising the step of administering to thesubject a therapeutically- or prophylactically-effective amount of anCFXTEN wherein said administration results in the improvement of one ormore biochemical or physiological parameters or clinical endpointsassociated with the coagulation factor-related disease, disorder orcondition. In one embodiment of the foregoing, the CFXTEN comprises aFVII. In another embodiment of the foregoing, the CFXTEN comprises aFIX. The effective amount produces a beneficial effect in helping totreat (e.g., cure or reduce the severity) or prevent (e.g., reduce thelikelihood of onset or severity) a coagulation factor-related disease,disorder or condition. As used herein, “treating” means administering adrug or a biologic (e.g., a CFXTEN) to achieve an improvement in anexisting disease, disorder or condition or preventing the occurrence ofa disease, disorder or condition (including prophylaxis). Atherapeutically-effective amount of a CFXTEN fusion protein can be thatamount of composition that, when administered as a single or as repeateddoses to a subject, leads to improvements in or amelioration of theunderlying disease, disorder or condition, or improvements in signs orsymptoms or physiologic parameters associated with the underlyingdisease, disorder or condition.

Hemostasis is regulated by multiple protein factors, and such proteins,as well as analogues thereof, have found utility in the treatment ofcoagulation factor-related diseases, disorders and conditions. However,the use of commercially-available coagulation factors has met with lessthan optimal success in the management of subjects afflicted with suchdiseases, disorders and conditions. In particular, dose optimization andfrequency of dosing is important for coagulation factors used in thetreatment or prevention of bleeding episodes in coagulationfactor-related diseases, disorders, or conditions, or uncontrolledbleeding in subjects not suffering from hemophilia. The fact thatcoagulation factors have a short half-life necessitates frequent dosingin order to achieve clinical benefit, which results in difficulties inthe management of such patients.

The invention provides methods of treatment comprising administering aCFXTEN composition to a subject suffering from or at risk of developinga coagulation factor-related disease, disorder or condition, wherein theadministration results in the improvement of one or more biochemical orphysiological parameters or clinical endpoints associated with thecondition. In one embodiment, the method of treatment comprisesadministering a therapeutically-effective amount of an CFXTENcomposition to a subject suffering from hemophilia A wherein theadministration results in the improvement of one or more biochemical orphysiological parameters or clinical endpoints associated with thecondition. In another embodiment, the method of treatment comprisesadministering a therapeutically-effective amount of an CFXTENcomposition to a subject suffering from hemophilia B wherein theadministration results in the improvement of one or more biochemical orphysiological parameters or clinical endpoints associated with thecondition. In another embodiment, the method of treatment comprisesadministering a therapeutically-effective amount of an CFXTENcomposition to a subject suffering from factor VII deficiency whereinsaid administration results in the improvement of one or morebiochemical or physiological parameters or clinical endpoints associatedwith the condition. In another embodiment, the method of treatmentcomprises administering a therapeutically-effective amount of an CFXTENcomposition to a subject suffering from or at risk of developinguncontrolled bleeding wherein the administration results in theimprovement of one or more biochemical or physiological parameters orclinical endpoints associated with the condition. In most instances, theembodiments of the disclosed method of treatments utilizing a CFXTENcomprising a FVII are compositions in which the FVII has been activated;i.e., FVIIa. However, the invention also contemplates CFXTENcompositions in which the FVII has not been activated. Because of thecomparatively long-half life of CFXTEN comprising FVII, it is believedthat compositions comprising the inactive form of FVII that can beactivated by mammalian endogenous proteases (because they include one ormore cleavage sequences; e.g., the sequences of Table 7) or the fusionprotein undergoes autoactivation such that 1) a bolus quantity ofactivated form of FVII is available by activation via clotting proteinsof the intrinsic coagulation cascade that has been initiated; or 2) apersistent quantity of activated form of FVII is available by activationvia proteases that are persistently or transiently present in thecirculation; e.g., MMP-12, MMP-17, etc. Thus, the invention provides amethod of treatment for a subject with a coagulation factor-relateddisease, disorder or conditions comprising administration of a CFXTENcomprising a FVII variant (as described above) wherein the FVII is notactivated but has one or more cleavage sequences that, when cleaved byan endogenous protease, converts the FVII component to the activatedform. In one embodiment of the foregoing, the method utilizes a CFXTENcomposition that has a terminal half-life of at least about 12 h, or atleast about 24 h, or at least about 48 h, or at least about 48 h, or atleast about 96 h, or at least about 144 h, or at least about 160 h.Accordingly, the method represents a means to treat subjects withcertain forms of chronic coagulopathies with what is essentially a“prodrug” form of FVII.

In some embodiments, administration of the CFXTEN to a subject resultsin an improvement in one or more of the biochemical, physiologic, orclinical parameters that is of greater magnitude than that of thecorresponding CF component not linked to XTEN, determined using the sameassay or based on a measured clinical parameter. In other embodiments,administration of the CFXTEN to a subject using a therapeuticallyeffective dose regimen results in activity in one or more of thebiochemical, physiologic, or clinical parameters that is of longerduration than the activity of the corresponding CF component not linkedto XTEN, determined using that same assay or based on a measuredclinical parameter. In one embodiment, the administration of atherapeutically effective amount of a CFXTEN comprising a FVII to asubject results in a reduction in prothrombin time at about 2-7 daysafter administration of at least about 5%, or about 10%, or about 20%,or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, ormore in the subject compared to the prothrombin time in a subject at acomparable time after administration of a comparable amount of FVII notlinked to XTEN. In another embodiment, the administration of a CFXTENcomprising a FVII to a subject using a therapeutically effective amountresults in maintenance of prothrombin times within 30% of normal in thesubject for a period of time that is at least two-fold, or aboutthree-fold, or at least about four-fold longer compared to a comparabledose regimen of FVII not linked to XTEN administered to a subject. Inanother embodiment, the administration of a therapeutically effectiveamount of a CFXTEN comprising a FIX to a subject results in a reductionin the activated partial prothrombin time at about 2-7 days afteradministration of at least about 5%, or about 10%, or about 20%, orabout 30%, or about 40%, or about 50%, or about 60%, or about 70%, ormore in the subject compared to the activated partial prothrombin timein a subject at a comparable time after administration of a comparableamount of FIX not linked to XTEN. In another embodiment, theadministration of a CFXTEN comprising a FIX to a subject using atherapeutically effective amount results in maintenance of activatedpartial prothrombin times within 30% of normal in the subject for aperiod of time that is at least two-fold, or at least about three-fold,or at least about four-fold longer compared to a comparable dose regimenof FIX not linked to XTEN administered to a subject. In anotherembodiment, the administration of a CFXTEN comprising a FVII to asubject using a therapeutically effective amount results in maintenanceof a bleeding time (in a bleeding time assay) within 30% of normal inthe subject for a period of time that is at least two-fold, or aboutthree-fold, or at least about four-fold longer compared to a comparableamount of FVII not linked to XTEN administered to a subject. In anotherembodiment, the administration of a CFXTEN comprising a FIX to a subjectusing a therapeutically effective amount results in maintenance of ableeding time (in a bleeding time assay) within 30% of normal in thesubject for a period of time that is at least two-fold, or aboutthree-fold, or at least about four-fold longer compared to a comparableamount of FIX not linked to XTEN administered to a subject.

As a result of the enhanced PK parameters of CFXTEN, as describedherein, the CF is administered using longer intervals between dosescompared to the corresponding CF not linked to XTEN to prevent, treat,alleviate, reverse or ameliorate symptoms or clinical abnormalities ofthe coagulation factor-related disease, disorder or condition or prolongthe survival of the subject being treated. In a particular application,CFXTEN comprising FVII have utility in the treatment of hemophilia A andhemophilia B.

It has been observed that FVIIa administered in high concentrations canfunction as a bypassing agent resulting in the activation of FX even inthe absence of FIX or FVIII. In order to act as a bypassing agent FVIIahas to be dosed at concentrations that exceed the level of FVIIa inhealthy people by approximately 100-fold. These levels are generallysafe because FVIIa has low activity in the absence of tissue factor(TF), to which FVII binds. Tissue factor is released or presented oninjured tissues which triggers clotting via the extrinsic system. Thecirculation half-life of FVIIa is in part limited by its inactivation byantithrombin (AT). Antithrombin can not bind to FVII but only to FVIIa.Thus, in one embodiment, the invention provides a method of treatinghemophilia A or B by administering an amount of CFXTEN comprising anactivated form of FVII, wherein the ability to activate FX in thecirculation of a subject is maintained for a period that is at leastabout two-fold longer, or at least about three-fold, or at least aboutfour-fold, or at least about five-fold, or at least about 10-fold, or atleast about 20-fold longer compared to FVII not linked to XTEN andadministered to a comparable subject at a comparable dose. The currentinvention further provides CFXTEN fusion proteins comprising FVII linkedto XTEN that can not be inactivated by AT by more than about 5% prior toits activation to FVIIa-XTEN. In one embodiment, the invention providesa method of treatment comprising administering a CFXTEN with a FVIIcomponent that is not activated, wherein the CFXTEN serves as acirculating depot wherein the area under the curve for the FVII that isactivated to FVIIa and not complexed with AT is at least about two-foldgreater, or at least about three-fold, or at least about four-fold, orat least about five-fold, or at least about 10-fold, or at least about20-fold greater than a FVII not linked to XTEN and administered at acomparable dose.

In some embodiments of the method of treatment, (i) a smaller molaramount of (e.g. of about two-fold less, or about three-fold less, orabout four-fold less, or about five-fold less, or about six-fold less,or about eight-fold less, or about 10-fold-less or greater) the fusionprotein is administered in comparison to the corresponding CF not linkedto the XTEN under an otherwise same dose regimen, and the fusion proteinachieves a comparable therapeutic effect as the corresponding CF notlinked to the XTEN; (ii) the fusion protein is administered lessfrequently (e.g., every two days, about every seven days, about every 14days, about every 21 days, or about, monthly) in comparison to thecorresponding CF not linked to the XTEN under an otherwise same doseamount, and the fusion protein achieves a comparable therapeutic effectas the corresponding CF not linked to the XTEN; or (iii) an accumulativesmaller molar amount (e.g. about 5%, or about 10%, or about 20%, orabout 40%, or about 50%, or about 60%, or about 70%, or about 80%, orabout 90% less) of the fusion protein is administered in comparison tothe corresponding CF not linked to the XTEN under the otherwise samedose regimen the fusion protein achieves a comparable therapeutic effectas the corresponding CF not linked to the XTEN. The accumulative smallermolar amount is measure for a period of at least about one week, orabout 14 days, or about 21 days, or about one month. The therapeuticeffect can be determined by any of the measured parameters or clinicalendpoints described herein.

The methods of the invention includes administration of consecutivedoses of a therapeutically effective amount of the CFXTEN for a periodof time sufficient to achieve and/or maintain the desired parameter orclinical effect, and such consecutive doses of a therapeuticallyeffective amount establishes the therapeutically effective dose regimenfor the CFXTEN, i.e., the schedule for consecutively administered dosesof the fusion protein composition, wherein the doses are given intherapeutically effective amounts to result in a sustained beneficialeffect on any clinical sign or symptom, aspect, measured parameter orcharacteristic of a coagulation factor-related disease state orcondition, including, but not limited to, those described herein. In oneembodiment, the method comprises administering atherapeutically-effective amount of a pharmaceutical compositioncomprising a CFXTEN fusion protein composition comprising a CF linked toan XTEN sequence(s) and at least one pharmaceutically acceptable carrierto a subject in need thereof that results in greater improvement in atleast one parameter, physiologic condition, or clinical outcome mediatedby the CF component(s) (non-limiting examples of which are describedabove) compared to the effect mediated by administration of apharmaceutical composition comprising a CF not linked to XTEN andadministered at a comparable dose. In one embodiment, the pharmaceuticalcomposition is administered at a therapeutically effective dose. Inanother embodiment, the pharmaceutical composition is administered usingmultiple consecutive doses using a therapeutically effective doseregimen (as defined herein) for the length of the dosing period.

A therapeutically effective amount of the CFXTEN varies according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the administered fusion protein to elicita desired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the CFXTEN areoutweighed by the therapeutically beneficial effects. A prophylacticallyeffective amount refers to an amount of CFXTEN required for the periodof time necessary to achieve the desired prophylactic result; e.g.,delayed onset of a bleeding episode. In the methods of treatment, thedose of the CFXTEN that is administered to a subject ranges from about0.5 mg to 1000 mg/dose, or from about 1 mg to 400 mg/dose, or from about10 mg to about 300 mg/dose for a 70 kg subject as loading andmaintenance doses, depending on the weight of the subject and theseverity of the condition.

The method of treatment comprises administration of a CFXTEN using atherapeutically effective dose regimen to effect improvements in one ormore parameters associated with coagulation factor diseases, disordersor conditions. In some embodiments, administration of the CFXTEN to asubject results in an improvement in one or more of the biochemical,physiologic, or clinical parameters that is of greater magnitude thanthat of the corresponding CF component not linked to XTEN, determinedusing the same assay or based on a measured clinical parameter. In otherembodiments, administration of the CFXTEN to a subject using atherapeutically effective dose regimen results in activity in one ormore of the biochemical, physiologic, or clinical parameters that is oflonger duration than the activity of one of the single CF components notlinked to XTEN, determined using that same assay or based on a measuredclinical parameter. In one embodiment of the foregoing, theadministration of the CFXTEN to a subject using a therapeuticallyeffective dose regimen results in an improvement in prothrombin time oractivated partial thromboplastin time of at least about 10%, or about20%, or about 30%, or about 40%, or about 50%, or about 60%, or about70%, or about 80%, or about 90%, or about 100% or more in the subjectcompared to a comparable dose of CF not linked to XTEN administered to asubject. In another embodiment of the foregoing, the administration ofthe CFXTEN to a subject using a therapeutically effective dose regimenresults in decreased instances of bleeding in the subject of at leastabout 10%, or about 20%, or about 30%, or about 40%, or about 50% ormore compared to a comparable dose regimen of CF not linked to XTENadministered to a subject.

The invention further contemplates that CFXTEN used in accordance withthe methods provided herein is administered in conjunction with othertreatment methods and compositions (e.g., other coagulation proteins)useful for treating coagulation factor-related diseases, disorders, andconditions, or conditions for which coagulation factor is adjunctivetherapy; e.g., bleeding episodes due to injury or surgery.

In another aspect, the invention provides a method of designing theCFXTEN compositions with desired pharmacologic or pharmaceuticalproperties. The CFXTEN fusion proteins are designed and prepared withvarious objectives in mind (compared to the CF components not linked tothe fusion protein), including improving the therapeutic efficacy forthe treatment of coagulation factor-related diseases, disorders, andconditions, enhancing the pharmacokinetic characteristics of the fusionproteins compared to the CF, lowering the dose or frequency of dosingrequired to achieve a pharmacologic effect, enhancing the pharmaceuticalproperties, and to enhance the ability of the CF components to remainwithin the therapeutic window for an extended period of time.

In general, the steps in the design and production of the fusionproteins and the inventive compositions, as illustrated in FIGS. 31-33,include: (1) the selection of CFs (e.g., native proteins, sequences ofTables 1 and 2, analogs or derivatives with activity) to treat theparticular disease, disorder or condition; (2) selecting the XTEN thatwill confer the desired PK and physicochemical characteristics on theresulting CFXTEN (e.g., the administration of the CFXTEN composition toa subject results in the fusion protein being maintained within thetherapeutic window for a greater period compared to CF not linked toXTEN); (3) establishing a desired N- to C-terminus configuration of theCFXTEN to achieve the desired efficacy or PK parameters; (4)establishing the design of the expression vector encoding the configuredCFXTEN; (5) transforming a suitable host with the expression vector; and(6) expression and recovery of the resultant fusion protein. For thoseCFXTEN for which an increase in half-life (greater than 24 h) or anincreased period of time spent within a therapeutic window is desired,the XTEN chosen for incorporation generally has at least about 100, orabout 144, or about 288, or about 432, or about 576, or about 864, orabout 875, or about 912, or about 923 amino acid residues where a singleXTEN is to be incorporated into the CFXTEN. In another embodiment, theCFXTEN comprises a first XTEN of the foregoing lengths, and at least asecond XTEN of about 36, or about 72, or about 144, or about 288, orabout 576, or about 864, or about 875, or about 912, or about 923 aminoacid residues.

In other embodiments, where an increase in half-life is not required,but an increase in a pharmaceutical property (e.g., solubility) isdesired, a CFXTEN is designed to include XTEN of shorter lengths. Insome embodiments of the foregoing, the CFXTEN comprises a CF linked toan XTEN having at least about 24, or about 36, or about 48, or about 60,or about 72, or about 84, or about 96 amino acid residues, in which thesolubility of the fusion protein under physiologic conditions is atleast three-fold greater than the corresponding CF not linked to XTEN,or alternatively, at least four-fold, or five-fold, or six-fold, orseven-fold, or eight-fold, or nine-fold, or at least 10-fold, or atleast 20-fold, or at least 30-fold, or at least 50-fold, or at least60-fold or greater than CF not linked to XTEN. In one embodiment of theforegoing, the CF is factor IX. In another embodiment, the CF is factorVII. In another embodiment, the XTEN is a sequence with at least about80%, or about 90%, or about 95% sequence identity compared to a sequencefrom Tables 4, and 9-13.

In another aspect, the invention provides methods of making CFXTENcompositions to improve ease of manufacture, result in increasedstability, increased water solubility, and/or ease of formulation, ascompared to the native CF. In one embodiment, the invention includes amethod of increasing the water solubility of a CF comprising the step oflinking the CF to one or more XTEN such that a higher concentration insoluble form of the resulting CFXTEN can be achieved, under physiologicconditions, compared to the CF in an un-fused state. Factors thatcontribute to the property of XTEN to confer increased water solubilityof CFs when incorporated into a fusion protein include the highsolubility of the XTEN fusion partner and the low degree ofself-aggregation between molecules of XTEN in solution. In someembodiments, the method results in a CFXTEN fusion protein wherein thewater solubility is at least about 20%, or at least about 30% greater,or at least about 50% greater, or at least about 75% greater, or atleast about 90% greater, or at least about 100% greater, or at leastabout 150% greater, or at least about 200% greater, or at least about400% greater, or at least about 600% greater, or at least about 800%greater, or at least about 1000% greater, or at least about 2000%greater, or at least about 4000% greater, or at least about 6000%greater under physiologic conditions, compared to the un-fused CF. Inone embodiment, the XTEN of the CFXTEN fusion protein is a sequence withat least about 80%, or about 90%, or about 95% sequence identitycompared to a sequence from Tables 4, and 9-13.

In another embodiment, the invention includes a method of increasing theshelf-life of a CF comprising the step of linking the CF with one ormore XTEN selected such that the shelf-life of the resulting CFXTEN isextended compared to the CF in an un-fused state. As used herein,shelf-life refers to the period of time over which the functionalactivity of a CF or CFXTEN that is in solution or in some other storageformulation remains stable without undue loss of activity. As usedherein, “functional activity” refers to a pharmacologic effect orbiological activity, such as the ability to bind a receptor or ligand,or substrate, or an enzymatic activity, or to display one or more knownfunctional activities associated with a CF, as known in the art. A CFthat degrades or aggregates generally has reduced functional activity orreduced bioavailability compared to one that remains in solution.Factors that contribute to the ability of the method to extend the shelflife of CFs when incorporated into a fusion protein include increasedwater solubility, reduced self-aggregation in solution, and increasedheat stability of the XTEN fusion partner. In particular, the lowtendency of XTEN to aggregate facilitates methods of formulatingpharmaceutical preparations containing higher drug concentrations ofCFs, and the heat-stability of XTEN contributes to the property ofCFXTEN fusion proteins to remain soluble and functionally active forextended periods. In one embodiment, the method results in CFXTEN fusionproteins with “prolonged” or “extended” shelf-life that exhibit greateractivity relative to a standard that has been subjected to the samestorage and handling conditions. The standard may be the un-fusedfull-length CF. In one embodiment, the method includes the step offormulating the isolated CFXTEN with one or more pharmaceuticallyacceptable excipients that enhance the ability of the XTEN to retain itsunstructured conformation and for the CFXTEN to remain soluble in theformulation for a time that is greater than that of the correspondingun-fused CF. In one embodiment, the method comprises linking a CF to oneor more XTEN selected from Tables 4 and 9-13 to create a CFXTEN fusionprotein results in a solution that retains greater than about 100% ofthe functional activity, or greater than about 105%, 110%, 120%, 130%,150% or 200% of the functional activity of a standard when compared at agiven time point and when subjected to the same storage and handlingconditions as the standard, thereby increasing its shelf-life.

Shelf-life may also be assessed in terms of functional activityremaining after storage, normalized to functional activity when storagebegan. CFXTEN fusion proteins of the invention with prolonged orextended shelf-life as exhibited by prolonged or extended functionalactivity retain about 50% more functional activity, or about 60%, 70%,80%, or 90% more of the functional activity of the equivalent CF notlinked to XTEN when subjected to the same conditions for the same periodof time. For example, a CFXTEN fusion protein of the inventioncomprising coagulation factor fused to one or more XTEN sequencesselected from Tables 4 and 9-13 retains about 80% or more of itsoriginal activity in solution for periods of up to 2 weeks, or 4 weeks,or 6 weeks or longer under various temperature conditions. In someembodiments, the CFXTEN retains at least about 50%, or about 60%, or atleast about 70%, or at least about 80%, and most preferably at leastabout 90% or more of its original activity in solution when heated at80° C. for 10 min. In other embodiments, the CFXTEN retains at leastabout 50%, preferably at least about 60%, or at least about 70%, or atleast about 80%, or alternatively at least about 90% or more of itsoriginal activity in solution when heated or maintained at 37° C. forabout 7 days. In another embodiment, CFXTEN fusion protein retains atleast about 80% or more of its functional activity after exposure to atemperature of about 30° C. to about 70° C. over a period of time ofabout one hour to about 18 hours. In the foregoing embodimentshereinabove described in this paragraph, the retained activity of theCFXTEN is at least about two-fold, or at least about three-fold, or atleast about four-fold, or at least about five-fold, or at least aboutsix-fold greater at a given time point than that of the corresponding CFnot linked to the fusion protein.

VII). The Nucleic Acids Sequences of the Invention

The present invention provides isolated polynucleic acids encodingCFXTEN chimeric fusion proteins and sequences complementary topolynucleic acid molecules encoding CFXTEN chimeric fusion proteins,including homologous variants thereof. In another aspect, the inventionencompasses methods to produce polynucleic acids encoding CFXTENchimeric fusion proteins and sequences complementary to polynucleic acidmolecules encoding CFXTEN chimeric fusion protein, including homologousvariants thereof. In general, and as illustrated in FIGS. 4-6, themethods of producing a polynucleotide sequence coding for a CFXTENfusion protein and expressing the resulting gene product includeassembling nucleotides encoding CF and XTEN, ligating the components inframe, incorporating the encoding gene into an expression vectorappropriate for a host cell, transforming the appropriate host cell withthe expression vector, and culturing the host cell under conditionscausing or permitting the fusion protein to be expressed in thetransformed host cell, thereby producing the biologically-active CFXTENpolypeptide, which is recovered as an isolated fusion protein bystandard protein purification methods known in the art. Standardrecombinant techniques in molecular biology is used to make thepolynucleotides and expression vectors of the present invention.

In accordance with the invention, nucleic acid sequences that encodeCFXTEN (or its complement) is used to generate recombinant DNA moleculesthat direct the expression of CFXTEN fusion proteins in appropriate hostcells. Several cloning strategies are suitable for performing thepresent invention, many of which is used to generate a construct thatcomprises a gene coding for a fusion protein of the CFXTEN compositionof the present invention, or its complement. In some embodiments, thecloning strategy is used to create a gene that encodes a monomericCFXTEN that comprises at least a first CF and at least a first XTENpolypeptide, or their complement. In one embodiment of the foregoing,the gene comprises a sequence encoding a CF or sequence variant. Inother embodiments, the cloning strategy is used to create a gene thatencodes a monomeric CFXTEN that comprises nucleotides encoding at leasta first molecule of CF or its complement and a first and at least asecond XTEN or their complement that is used to transform a host cellfor expression of the fusion protein of the CFXTEN composition. In theforegoing embodiments hereinabove described in this paragraph, the genescan further comprise nucleotides encoding spacer sequences that alsoencode cleavage sequence(s).

In designing a desired XTEN sequences, it was discovered that thenon-repetitive nature of the XTEN of the inventive compositions isachieved despite use of a “building block” molecular approach in thecreation of the XTEN-encoding sequences. This was achieved by the use ofa library of polynucleotides encoding peptide sequence motifs, describedabove, that are then ligated and/or multimerized to create the genesencoding the XTEN sequences (see FIGS. 4 and 5 and Examples). Thus,while the XTEN(s) of the expressed fusion protein may consist ofmultiple units of as few as four different sequence motifs, because themotifs themselves consist of non-repetitive amino acid sequences, theoverall XTEN sequence is rendered non-repetitive. Accordingly, in oneembodiment, the XTEN-encoding polynucleotides comprise multiplepolynucleotides that encode non-repetitive sequences, or motifs,operably linked in frame and in which the resulting expressed XTEN aminoacid sequences are non-repetitive.

In one approach, a construct is first prepared containing the DNAsequence corresponding to CFXTEN fusion protein. DNA encoding the CF ofthe compositions is obtained from a cDNA library prepared using standardmethods from tissue or isolated cells believed to possess CF mRNA and toexpress it at a detectable level. Libraries is screened with probescontaining, for example, about 20 to 100 bases designed to identify theCF gene of interest by hybridization using conventional molecularbiology techniques. The best candidates for probes are those thatrepresent sequences that are highly homologous for coagulation factor,and should be of sufficient length and sufficiently unambiguous thatfalse positives are minimized, but may be degenerate at one or morepositions. If necessary, the coding sequence can be obtained usingconventional primer extension procedures as described in Sambrook, etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA. One can then usepolymerase chain reaction (PCR) methodology to amplify the target DNA orRNA coding sequence to obtain sufficient material for the preparation ofthe CFXTEN constructs containing the CF gene(s). Assays can then beconducted to confirm that hybridizing full-length genes are the desiredCF gene(s). By these conventional methods, DNA can be convenientlyobtained from a cDNA library prepared from such sources. The CF encodinggene(s) is also be obtained from a genomic library or created bystandard synthetic procedures known in the art (e.g., automated nucleicacid synthesis using, for example one of the methods described in Engelset al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNAsequences obtained from publicly available databases, patents, orliterature references. Such procedures are well known in the art andwell described in the scientific and patent literature. For example,sequences can be obtained from Chemical Abstracts Services (CAS)Registry Numbers (published by the American Chemical Society) and/orGenBank Accession Numbers (e.g., Locus ID, NP_XXXXX, and XP_XXXXX) ModelProtein identifiers available through the National Center forBiotechnology Information (NCBI) webpage, available on the world wideweb at ncbi.nlm.nih.gov that correspond to entries in the CAS Registryor GenBank database that contain an amino acid sequence of the proteinof interest or of a fragment or variant of the protein. For suchsequence identifiers provided herein, the summary pages associated witheach of these CAS and GenBank and GenSeq Accession Numbers as well asthe cited journal publications (e.g., PubMed ID number (PMID)) are eachincorporated by reference in their entireties, particularly with respectto the amino acid sequences described therein. In one embodiment, the CFencoding gene encodes a protein from any one of Table 1 or Table 2, or afragment or variant thereof.

A gene or polynucleotide encoding the CF portion of the subject CFXTENprotein, in the case of an expressed fusion protein that comprises asingle CF is then be cloned into a construct, which is a plasmid orother vector under control of appropriate transcription and translationsequences for high level protein expression in a biological system. In alater step, a second gene or polynucleotide coding for the XTEN isgenetically fused to the nucleotides encoding the N- and/or C-terminusof the CF gene by cloning it into the construct adjacent and in framewith the gene(s) coding for the CF. This second step occurs through aligation or multimerization step. In the foregoing embodimentshereinabove described in this paragraph, it is to be understood that thegene constructs that are created can alternatively be the complement ofthe respective genes that encode the respective fusion proteins.

The gene encoding for the XTEN can be made in one or more steps, eitherfully synthetically or by synthesis combined with enzymatic processes,such as restriction enzyme-mediated cloning, PCR and overlap extension,including methods more fully described in the Examples. The methodsdisclosed herein can be used, for example, to ligate short sequences ofpolynucleotides encoding XTEN into longer XTEN genes of a desired lengthand sequence. In one embodiment, the method ligates two or morecodon-optimized oligonucleotides encoding XTEN motif or segmentsequences of about 9 to 14 amino acids, or about 12 to 20 amino acids,or about 18 to 36 amino acids, or about 48 to about 144 amino acids, orabout 144 to about 288 or longer, or any combination of the foregoingranges of motif or segment lengths.

Alternatively, the disclosed method is used to multimerize XTEN-encodingsequences into longer sequences of a desired length; e.g., a geneencoding 36 amino acids of XTEN can be dimerized into a gene encoding 72amino acids, then 144, then 288, etc. Even with multimerization, XTENpolypeptides can be constructed such that the XTEN-encoding gene has lowor virtually no repetitiveness through design of the codons selected forthe motifs of the shortest unit being used, which can reducerecombination and increase stability of the encoding gene in thetransformed host. Genes encoding XTEN with non-repetitive sequences isassembled from oligonucleotides using standard techniques of genesynthesis. The gene design can be performed using algorithms thatoptimize codon usage and amino acid composition. In one method of theinvention, a library of relatively short XTEN-encoding polynucleotideconstructs is created and then assembled, as illustrated in FIGS. 4 and5. This can be a pure codon library such that each library member hasthe same amino acid sequence but many different coding sequences arepossible. Such libraries can be assembled from partially randomizedoligonucleotides and used to generate large libraries of XTEN segmentscomprising the sequence motifs. The randomization scheme can beoptimized to control amino acid choices for each position as well ascodon usage. Exemplary methods to achieve the foregoing are disclosed inthe Examples.

Polynucleotide Libraries

In another aspect, the invention provides libraries of polynucleotidesthat encode XTEN sequences that are used to assemble genes that encodeXTEN of a desired length and sequence.

In certain embodiments, the XTEN-encoding library constructs comprisepolynucleotides that encode polypeptide segments of a fixed length. Asan initial step, a library of oligonucleotides that encode motifs of9-14 amino acid residues can be assembled. In a preferred embodiment,libraries of oligonucleotides that encode motifs of 12 amino acids areassembled.

The XTEN-encoding sequence segments can be dimerized or multimerizedinto longer encoding sequences. Dimerization or multimerization can beperformed by ligation, overlap extension, PCR assembly or similarcloning techniques known in the art. This process of can be repeatedmultiple times until the resulting XTEN-encoding sequences have reachedthe organization of sequence and desired length, providing theXTEN-encoding genes. As will be appreciated, a library ofpolynucleotides that encodes, e.g., 12 amino acid motifs can bedimerized and/or ligated into a library of polynucleotides that encode36 amino acids. Libraries encoding motifs of different lengths; e.g.,9-14 amino acid motifs leading to libraries encoding 27 to 42 aminoacids are contemplated by the invention. In turn, the library ofpolynucleotides that encode 27 to 42 amino acids, and preferably 36amino acids (as described in the Examples) can be serially dimerizedinto a library containing successively longer lengths of polynucleotidesthat encode XTEN sequences of a desired length for incorporation intothe gene encoding the CFXTEN fusion protein, as disclosed herein. Insome embodiments, libraries are assembled of polynucleotides that encodeamino acids that are limited to specific sequence XTEN families; e.g.,AD, AE, AF, AG, AM, or AQ sequences of Table 3. In other embodiments,libraries comprise sequences that encode two or more of the motif familysequences from Table 3. The names and sequences of representative,non-limiting polynucleotide sequences of libraries that encode 36mersare presented in Tables 9-12, and the methods used to create them aredescribed more fully in the respective Examples. In other embodiments,libraries that encode XTEN are constructed from segments ofpolynucleotide codons linked in a randomized sequence that encode aminoacids wherein at least about 80%, or at least about 90%, or at leastabout 91%, or at least about 92%, or at least about 93%, or at leastabout 94%, or at least about 95%, or at least about 97%, or at leastabout 98%, or at least about 99% of the codons are selected from thegroup consisting of condons for glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P) amino acids. The librariescan be used, in turn, for serial dimerization or ligation to achievepolynucleotide sequence libraries that encode XTEN sequences, forexample, of 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids,or up to a total length of about 3000 amino acids, as well asintermediate lengths, in which the encoded XTEN can have one or more ofthe properties disclosed herein, when expressed as a component of aCFXTEN fusion protein. In some cases, the polynucleotide librarysequences may also include additional bases used as “sequencingislands,” described more fully below.

FIG. 32 is a schematic flowchart of representative, non-limiting stepsin the assembly of a XTEN polynucleotide construct and a CFXTENpolynucleotide construct in the embodiments of the invention. Individualoligonucleotides 501 are annealed into sequence motifs 502 such as a 12amino acid motif (“12-mer”), which is subsequently ligated with an oligocontaining BbsI, and KpnI restriction sites 503. Additional sequencemotifs from a library are annealed to the 12-mer until the desiredlength of the XTEN gene 504 is achieved. The XTEN gene is cloned into astuffer vector. The vector optionally encodes a Flag sequence 506followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnIsites 507 and, in this case, a single CF gene (encoding FIX in thisexample) 508, resulting in the gene encoding a CFXTEN comprising asingle CF 500. A non-exhaustive list of the XTEN names forpolynucleotides encoding XTEN and precursor sequences is provided inTable 8.

TABLE 8 DNA sequences of XTEN and precursor sequences SEQ XTEN ID NameNO: DNA Nucleotide Sequence AE48 111ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCT AM48 112ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCT AE144 113GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCAGGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA AF144 114GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCA AE288 115GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AE576 116GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC A AF576 117GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA AE624 118ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA AM875 119GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA AE864 120GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACC A AF864 121GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAXXXXXXXXTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAXXXX was inserted in two areas where no sequenceinformation is available. AG864 122GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTAC CGGTTCTCCA AM923123 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA AE912 124ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AM1318 125GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA BC864 126GGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGCGCATCCGAGCCTACCTCTACTGAACCAGGTAGCGAACCGGCTACCTCCGGTACTGAGCCATCAGGTAGCGAACCGGCAACTTCCGGTACTGAACCATCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGCAGGTAGCGGCGCATCCGAACCTACTTCCACTGAACCAGGTACTAGCGAGCCATCCACCTCTGAACCAGGTGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAACCGGCTACTTCCGGCACTGAACCATCAGGTAGCGAACCAGCAACCTCCGGTACTGAACCATCAGGTACTTCCACTGAACCATCCGAACCGGGTAGCGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTAGCGGCGCATCTGAGCCTACTTCCACTGAACCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTAGCGAACCATCCACCTCCGAACCAGGCGCAGGTAGCGGTGCATCTGAACCGACTTCTACTGAACCAGGTACTTCCACTGAACCATCTGAGCCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGC AGCGCA BD864 127GGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTTCCACTGAAGCAAGTGAAGGCTCCGCATCAGGTACTTCCACCGAAGCAAGCGAAGGCTCCGCATCAGGTACTAGTGAGTCCGCAACTAGCGAATCCGGTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCAGGTTCTGAGACTTCCACCGAAGCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCCGCATCAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTTCCACCGAAGCAAGCGAAGGTTCCGCATCAGGTACTTCCACCGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACTGCTACTTCCGGCTCCGAGACTGCAGGTAGCGAAACTGCTACTTCTGGCTCCGAAACTGCAGGTACTTCTACTGAGGCTAGTGAAGGTTCCGCATCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGAC TGCA

One may clone the library of XTEN-encoding genes into one or moreexpression vectors known in the art. To facilitate the identification ofwell-expressing library members, one can construct the library as fusionto a reporter protein. Non-limiting examples of suitable reporter genesare green fluorescent protein, luciferace, alkaline phosphatase, andbeta-galactosidase. By screening, one can identify short XTEN sequencesthat can be expressed in high concentration in the host organism ofchoice. Subsequently, one can generate a library of random XTEN dimersand repeat the screen for high level of expression. Subsequently, onecan screen the resulting constructs for a number of properties such aslevel of expression, protease stability, or binding to antiserum.

One aspect of the invention is to provide polynucleotide sequencesencoding the components of the fusion protein wherein the creation ofthe sequence has undergone codon optimization. Of particular interest iscodon optimization with the goal of improving expression of thepolypeptide compositions and to improve the genetic stability of theencoding gene in the production hosts. For example, codon optimizationis of particular importance for XTEN sequences that are rich in glycineor that have very repetitive amino acid sequences. Codon optimization isperformed using computer programs (Gustafsson, C., et al. (2004) TrendsBiotechnol, 22: 346-53), some of which minimize ribosomal pausing (CodaGenomics Inc.). In one embodiment, one can perform codon optimization byconstructing codon libraries where all members of the library encode thesame amino acid sequence but where codon usage is varied. Such librariescan be screened for highly expressing and genetically stable membersthat are particularly suitable for the large-scale production ofXTEN-containing products. When designing XTEN sequences one can considera number of properties. One can minimize the repetitiveness in theencoding DNA sequences. In addition, one can avoid or minimize the useof codons that are rarely used by the production host (e.g. the AGG andAGA arginine codons and one leucine codon in E. coli). In the case of E.coli, two glycine codons, GGA and GGG, are rarely used in highlyexpressed proteins. Thus codon optimization of the gene encoding XTENsequences can be very desirable. DNA sequences that have a high level ofglycine tend to have a high GC content that can lead to instability orlow expression levels. Thus, when possible, it is preferred to choosecodons such that the GC-content of XTEN-encoding sequence is suitablefor the production organism that will be used to manufacture the XTEN.

Optionally, the full-length XTEN-encoding gene comprises one or moresequencing islands. In this context, sequencing islands areshort-stretch sequences that are distinct from the XTEN libraryconstruct sequences and that include a restriction site not present orexpected to be present in the full-length XTEN-encoding gene. In oneembodiment, a sequencing island is the sequence5′-AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGT-3′ (SEQ ID NO: 128). In anotherembodiment, a sequencing island is the sequence

(SEQ ID NO: 129) 5′-AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGT-3′.

In one embodiment, polynucleotide libraries are constructed using thedisclosed methods wherein all members of the library encode the sameamino acid sequence but where codon usage for the respective amino acidsin the sequence is varied. Such libraries can be screened for highlyexpressing and genetically stable members that are particularly suitablefor the large-scale production of XTEN-containing products.

Optionally, one can sequence clones in the library to eliminate isolatesthat contain undesirable sequences. The initial library of short XTENsequences allows some variation in amino acid sequence. For instance onecan randomize some codons such that a number of hydrophilic amino acidscan occur in a particular position. During the process of iterativemultimerization one can screen the resulting library members for othercharacteristics like solubility or protease resistance in addition to ascreen for high-level expression.

Once the gene that encodes the XTEN of desired length and properties isselected, it is genetically fused at the desired location to thenucleotides encoding the CF gene(s) by cloning it into the constructadjacent and in frame with the gene coding for CF, or alternativelybetween nucleotides encoding adjacent domains of the CF, oralternatively within a sequence encoding a given CF domain, oralternatively in frame with nucleotides encoding a spacer/cleavagesequence linked to a terminal XTEN. The invention provides variouspermutations of the foregoing, depending on the CFXTEN to be encoded.For example, a gene encoding a CFXTEN fusion protein comprising a CF andtwo XTEN, such as embodied by formula VI, as depicted above, the genewould have polynucleotides encoding CF, encoding two XTEN, which can beidentical or different in composition and sequence length. In onenon-limiting embodiment of the foregoing, the CF polynucleotides wouldencode coagulation factor and the polynucleotides encoding theC-terminus XTEN would encode AE864 and the polynucleotides encoding aninternal XTEN adjacent to the C-terminus of EGF2 would encode AE144. Thestep of cloning the CF genes into the XTEN construct can occur through aligation or multimerization step, as shown in FIG. 32. The constructsencoding CFXTEN fusion proteins can be designed in differentconfigurations of the components XTEN, CF, and spacer sequences, such asthe configurations of formulae I-VI. In one embodiment, the constructcomprises polynucleotide sequences complementary to, or those thatencode a monomeric polypeptide of components in the following order (5′to 3′) CF and XTEN. In another embodiment, the construct comprisespolynucleotide sequences complementary to, or those that encode amonomeric polypeptide of components in the following order (5′ to 3′)CF, spacer sequence, and XTEN. The spacer polynucleotides can optionallycomprise sequences encoding cleavage sequences. As will be apparent tothose of skill in the art, other permutations or multimers of theforegoing are possible.

The invention also encompasses polynucleotides comprising XTEN-encodingpolynucleotide variants that have a high percentage of sequence identitycompared to (a) a polynucleotide sequence from Table 8, or (b) sequencesthat are complementary to the polynucleotides of (a). A polynucleotidewith a high percentage of sequence identity is one that has at leastabout an 80% nucleic acid sequence identity, alternatively at leastabout 81%, alternatively at least about 82%, alternatively at leastabout 83%, alternatively at least about 84%, alternatively at leastabout 85%, alternatively at least about 86%, alternatively at leastabout 87%, alternatively at least about 88%, alternatively at leastabout 89%, alternatively at least about 90%, alternatively at leastabout 91%, alternatively at least about 92%, alternatively at leastabout 93%, alternatively at least about 94%, alternatively at leastabout 95%, alternatively at least about 96%, alternatively at leastabout 97%, alternatively at least about 98%, and alternatively at leastabout 99% nucleic acid sequence identity compared to (a) or (b) of theforegoing, or that can hybridize with the target polynucleotide or itscomplement under stringent conditions.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may also be determined conventionally by usingknown software or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics. 1981.2: 482-489), to find the best segment of identity or similarity betweentwo sequences. Gap performs global alignments: all of one sequence withall of another similar sequence using the method of Needleman andWunsch, (Journal of Molecular Biology. 1970. 48:443-453). When using asequence alignment program such as BestFit, to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores.

Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. As used herein, the term “complementarysequences” means nucleic acid sequences that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above, or as defined as being capable of hybridizing to thepolynucleotides that encode the CFXTEN sequences under stringentconditions, such as those described herein.

The resulting polynucleotides encoding the CFXTEN chimeric fusionproteins can then be individually cloned into an expression vector. Thenucleic acid sequence is inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Vectorcomponents generally include, but are not limited to, one or more of asignal sequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of thesecomponents employs standard ligation techniques which are known to theskilled artisan. Such techniques are well known in the art and welldescribed in the scientific and patent literature.

Various vectors are publicly available. The vector may, for example, bein the form of a plasmid, cosmid, viral particle, or phage that mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The invention provides for the use of plasmid vectors containingreplication and control sequences that are compatible with andrecognized by the host cell, and are operably linked to the CFXTEN genefor controlled expression of the CFXTEN fusion proteins. The vectorordinarily carries a replication site, as well as sequences that encodeproteins that are capable of providing phenotypic selection intransformed cells. Such vector sequences are well known for a variety ofbacteria, yeast, and viruses. Useful expression vectors that can be usedinclude, for example, segments of chromosomal, non-chromosomal andsynthetic DNA sequences. “Expression vector” refers to a DNA constructcontaining a DNA sequence that is operably linked to a suitable controlsequence capable of effecting the expression of the DNA encoding thefusion protein in a suitable host. The requirements are that the vectorsare replicable and viable in the host cell of choice. Low- or high-copynumber vectors may be used as desired.

Other suitable vectors include, but are not limited to, derivatives ofSV40 and pcDNA and known bacterial plasmids such as col EI, pCR1,pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40(1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAssuch as the numerous derivatives of phage I such as NM98 9, as well asother phage DNA such as M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2 micron plasmid or derivatives of the 2 mplasmid, as well as centomeric and integrative yeast shuttle vectors;vectors useful in eukaryotic cells such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orthe expression control sequences; and the like. Yeast expression systemsthat can also be used in the present invention include, but are notlimited to, the non-fusion pYES2 vector (Invitrogen), the fusionpYESHisA, B, C (Invitrogen), pRS vectors and the like.

The control sequences of the vector include a promoter to effecttranscription, an optional operator sequence to control suchtranscription, a sequence encoding suitable mRNA ribosome binding sites,and sequences that control termination of transcription and translation.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of theDNA encoding the CF polypeptide variant in mammalian cells are the SV40promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), theMT-1 (metallothionein gene) promoter (Palmiter et al., Science 222(1983), 809-814), the CMV promoter (Boshart et al., Cell 41:521-530,1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol.Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences suchas UCOE (ubiquitous chromatin opening elements).

Examples of suitable promoters for use in filamentous fungus host cellsare, for instance, the ADH3 promoter or the tpiA promoter. Examples ofother useful promoters are those derived from the gene encoding A.oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. niger or A.awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters.

Promoters suitable for use in expression vectors with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)], all is operably linked to the DNA encoding CFXTEN polypeptides.Promoters for use in bacterial systems can also contain a Shine-Dalgarno(S.D.) sequence, operably linked to the DNA encoding CFXTENpolypeptides.

The invention contemplates use of other expression systems including,for example, a baculovirus expression system with both non-fusiontransfer vectors, such as, but not limited to pVL941 Summers, et al.,Virology 84:390-402 (1978)), pVL1393 (Invitrogen), pVL1392 (Summers, etal., Virology 84:390-402 (1978) and Invitrogen) and pBlueBacIII(Invitrogen), and fusion transfer vectors such as, but not limited to,pAc7 00 (Summers, et al., Virology 84:390-402 (1978)), pAc701 andpAc70-2 (same as pAc700, with different reading frames), pAc360Invitrogen) and pBlueBacHisA, B, C (; Invitrogen) can be used.

Examples of suitable promoters for directing the transcription of theDNA encoding the CF polypeptide variant in mammalian cells are the CMVpromoter (Boshart et al., Cell 41:521-530, 1985), the SV40 promoter(Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1(metallothionein gene) promoter (Palmiter et al., Science 222 (1983),809-814), the adenovirus 2 major late promoter (Kaufman and Sharp, Mol.Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences suchas UCOE (ubiquitous chromatin opening elements).

Examples of suitable promoters for use in filamentous fungus host cellsare, for instance, the ADH3 promoter or the tpiA promoter.

The DNA sequences encoding the CFXTEN may also, if necessary, beoperably connected to a suitable terminator, such as the hGH terminator(Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp.419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099).Expression vectors may also contain a set of RNA splice sites locateddownstream from the promoter and upstream from the insertion site forthe CFXTEN sequence itself, including splice sites obtained fromadenovirus. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the hGHterminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981). Theexpression vectors may also include a noncoding viral leader sequence,such as the adenovirus 2 tripartite leader, located between the promoterand the RNA splice sites; and enhancer sequences, such as the SV40enhancer.

To direct the CFXTEN of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (a.k.a., a leadersequence, a prepro sequence, or a pre sequence) may be included in therecombinant vector. The secretory signal sequence is operably linked tothe DNA sequences encoding the CFXTEN, usually positioned 5′ to the DNAsequence encoding the CFXTEN fusion protein. The secretory signalsequence may be that, normally associated with the protein or may befrom a gene encoding another secreted protein. Non-limiting examplesinclude OmpA, PhoA, and DsbA for E. coli expression, ppL-alpha, DEX4,invertase signal peptide, acid phosphatase signal peptide, CPY, or INU1for yeast expression, and IL2L, SV40, IgG kappa and IgG lambda formammalian expression. Signal sequences are typically proteolyticallyremoved from the protein during the translocation and secretion process,generating a defined N-terminus. Methods are disclosed in Arnau, et al.,Protein Expression and Purification 48: 1-13 (2006).

The procedures used to ligate the DNA sequences coding for the CFXTEN,the promoter and optionally the terminator and/or secretory signalsequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

In other cases, the invention provides constructs and methods of makingconstructs comprising an polynucleotide sequence optimized forexpression that encodes at least about 20 to about 60 amino acids withXTEN characteristics that can be included at the N-terminus of an XTENcarrier encoding sequence (in other words, the polynucleotides encodingthe 20-60 encoded optimized amino acids are linked in frame topolynucleotides encoding an XTEN component that is N-terminal to CF) topromote the initiation of translation to allow for expression of XTENfusions at the N-terminus of proteins without the presence of a helperdomain. In an advantage of the foregoing, the sequence does not requiresubsequent cleavage, thereby reducing the number of steps to manufactureXTEN-containing compositions. As described in more detail in theExamples, the optimized N-terminal sequence has attributes of anunstructured protein, but may include nucleotide bases encoding aminoacids selected for their ability to promote initiation of translationand enhanced expression. In one embodiment of the foregoing, theoptimized polynucleotide encodes an XTEN sequence with at least about90% sequence identity compared to AE912. In another embodiment of theforegoing, the optimized polynucleotide encodes an XTEN sequence with atleast about 90% sequence identity compared to AM923. In anotherembodiment of the foregoing, the optimized polynucleotide encodes anXTEN sequence with at least about 90% sequence identity compared toAE48. In another embodiment of the foregoing, the optimizedpolynucleotide encodes an XTEN sequence with at least about 90% sequenceidentity compared to AM48. In one embodiment, the optimizedpolynucleotide NTS comprises a sequence that exhibits at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%, sequence identity compared to a sequence or itscomplement selected from

AE48: (SEQ ID NO: 130)5′-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA-3′ and AM48:(SEQ ID NO: 131) 5′-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA-3′

In this manner, a chimeric DNA molecule coding for a monomeric CFXTENfusion protein is generated within the construct. Optionally, thischimeric DNA molecule may be transferred or cloned into anotherconstruct that is a more appropriate expression vector. At this point, ahost cell capable of expressing the chimeric DNA molecule can betransformed with the chimeric DNA molecule.

Examples of mammalian cell lines for use in the present invention arethe COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10))and BHK-293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977), BHK-570 cells (ATCC CRL 10314), CHO-K1 (ATCC CCL 61), CHO-S(Invitrogen 11619-012), and 293-F (Invitrogen R790-7). A tk⁻ts13 BHKcell line is also available from the ATCC under accession number CRL1632. In addition, a number of other cell lines may be used within thepresent invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600),Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Humanlung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) andDUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220,1980).

Examples of suitable yeasts cells include cells of Saccharomyces spp. orSchizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidesthere from are described, e.g. in U.S. Pat. Nos. 4,599,311, 4,931,373,4,870,008, 5,037,743, and 4,845,075, all of which are herebyincorporated by reference. Transformed cells are selected by a phenotypedetermined by a selectable marker, commonly drug resistance or theability to grow in the absence of a particular nutrient, e.g. leucine. Apreferred vector for use in yeast is the POT1 vector disclosed in U.S.Pat. No. 4,931,373. The DNA sequences encoding the CFXTEN may bepreceded by a signal sequence and optionally a leader sequence, e.g. asdescribed above. Further examples of suitable yeast cells are strains ofKluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, orPichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132,1986, pp. 3459-3465; U.S. Pat. No. 4,882,279). Examples of other fungalcells are cells of filamentous fungi, e.g. Aspergillus spp., Neurosporaspp., Fusarium spp. or Trichoderma spp., in particular strains of A.oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for theexpression of proteins is described in, e.g., EP 272 277, EP 238 023, EP184 438 The transformation of E oxysporum may, for instance, be carriedout as described by Malardier et al., 1989, Gene 78: 147-156. Thetransformation of Trichoderma spp. may be performed for instance asdescribed in EP 244 234.

Other suitable cells that can be used in the present invention include,but are not limited to, prokaryotic host cells strains such asEscherichia coli, (e.g., strain DHS-a), Bacillus subtilis, Salmonellatyphimurium, or strains of the genera of Pseudomonas, Streptomyces andStaphylococcus. Non-limiting examples of suitable prokaryotes includethose from the genera: Actinoplanes; Archaeoglobus; Bdellovibrio;Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus;Listeria; Oceanobacillus; Paracoccus; Pseudomonas; Staphylococcus;Streptococcus; Streptomyces; Thermoplasma; and Vibrio.

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g., Kaufman and Sharp, J.Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet.1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982),422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973),transfection with many commercially available reagents such as FuGENEGRoche Diagnostics, Mannheim, Germany) or lipofectamine (Invitrogen) orby electroporation (Neumann et al., EMBO J. 1:841-845, 1982). Toidentify and select cells that express the exogenous DNA, a gene thatconfers a selectable phenotype (a selectable marker) is generallyintroduced into cells along with the gene or cDNA of interest. Preferredselectable markers include genes that confer resistance to drugs such asneomycin, hygromycin, puromycin, zeocin, and methotrexate. Theselectable marker may be an amplifiable selectable marker. A preferredamplifiable selectable marker is a dihydrofolate reductase (DHFR)sequence. Further examples of selectable markers are well known to oneof skill in the art and include reporters such as enhanced greenfluorescent protein (EGFP), beta-galactosidase (β-gal) orchloramphenicol acetyltransferase (CAT). Selectable markers are reviewedby Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham,Mass., incorporated herein by reference). The person skilled in the artwill easily be able to choose suitable selectable markers. Any knownselectable marker may be employed so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If, on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA,” to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term “appropriate growth medium” means amedium containing nutrients and other components required for the growthof cells and the expression of the CFXTEN of interest. Media generallyinclude a carbon source, a nitrogen source, essential amino acids,essential sugars, vitamins, salts, phospholipids, protein and growthfactors. For production of gamma-carboxylated proteins, the medium willcontain vitamin K, preferably at a concentration of about 0.1 μg/ml toabout 5 μg/ml. Drug selection is then applied to select for the growthof cells that are expressing the selectable marker in a stable fashion.For cells that have been transfected with an amplifiable selectablemarker the drug concentration may be increased to select for anincreased copy number of the cloned sequences, thereby increasingexpression levels. Clones of stably transfected cells are then screenedfor expression of the CF polypeptide variant of interest.

The transformed or transfected host cell is then cultured in a suitablenutrient medium under conditions permitting expression of the CFpolypeptide variant after which the resulting peptide may be recoveredfrom the culture. The medium used to culture the cells may be anyconventional medium suitable for growing the host cells, such as minimalor complex media containing appropriate supplements. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection). The culture conditions, such as temperature, pH and thelike, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Gene expression may be measured in a sample directly, for example, byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205(1980)], dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological offluorescent methods, such as immunohistochemical staining of cells ortissue sections and assay of cell culture or body fluids or thedetection of selectable markers, to quantitate directly the expressionof gene product. Antibodies useful for immunohistochemical stainingand/or assay of sample fluids may be either monoclonal or polyclonal,and may be prepared in any mammal. Conveniently, the antibodies may beprepared against a native sequence CF polypeptide or against a syntheticpeptide based on the DNA sequences provided herein or against exogenoussequence fused to CF and encoding a specific antibody epitope. Examplesof selectable markers are well known to one of skill in the art andinclude reporters such as enhanced green fluorescent protein (EGFP),beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).

Expressed CFXTEN polypeptide product(s) may be purified via methodsknown in the art or by methods disclosed herein. Procedures such as gelfiltration, affinity purification (e.g., using an anti-CF antibodycolumn), salt fractionation, ion exchange chromatography, size exclusionchromatography, hydroxyapatite adsorption chromatography, hydrophobicinteraction chromatography and gel electrophoresis may be used; eachtailored to recover and purify the fusion protein produced by therespective host cells. Additional purification may be achieved byconventional chemical purification means, such as high performanceliquid chromatography. Some expressed CFXTEN may require refoldingduring isolation and purification. Methods of purification are describedin Robert K. Scopes, Protein Purification: Principles and Practice,Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al.,supra. Multi-step purification separations are also described in Baron,et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J.Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it ispreferred that the CFXTEN fusion proteins of the invention aresubstantially pure. Thus, in a preferred embodiment of the invention theCFXTEN of the invention is purified to at least about 90 to 95%homogeneity, preferably to at least about 98% homogeneity. Purity may beassessed by, e.g., gel electrophoresis, HPLC, and amino-terminal aminoacid sequencing.

VIII). Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprisingCFXTEN. In one embodiment, the pharmaceutical composition comprises theCFXTEN fusion protein and at least one pharmaceutically acceptablecarrier. CFXTEN polypeptides of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the polypeptide is combined in admixture with apharmaceutically acceptable carrier vehicle, such as aqueous solutionsor buffers, pharmaceutically acceptable suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. Therapeutic formulations areprepared for storage by mixing the active ingredient having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers, as described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilizedformulations or aqueous solutions.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition, thepharmaceutical compositions can also contain other pharmaceuticallyactive compounds or a plurality of compounds of the invention.

More particularly, the present pharmaceutical compositions may beadministered for therapy by any suitable route including oral, rectal,nasal, topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, subcutaneous by infusionpump, intramuscular, intravenous and intradermal), intravitreal, andpulmonary. It will also be appreciated that the preferred route willvary with the condition and age of the recipient, and the disease beingtreated.

In one embodiment, the pharmaceutical composition is administeredsubcutaneously. In this embodiment, the composition may be supplied as alyophilized powder to be reconstituted prior to administration. Thecomposition may also be supplied in a liquid form, which can beadministered directly to a patient. In one embodiment, the compositionis supplied as a liquid in a pre-filled syringe such that a patient caneasily self-administer the composition.

Extended release formulations useful in the present invention may beoral formulations comprising a matrix and a coating composition.Suitable matrix materials may include waxes (e.g., camauba, bees wax,paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols),oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil,beef tallow, palm oil, and soya bean oil), and polymers (e.g.,hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and polyethylene glycol). Other suitable matrix tablettingmaterials are microcrystalline cellulose, powdered cellulose,hydroxypropyl cellulose, ethyl cellulose, with other carriers, andfillers. Tablets may also contain granulates, coated powders, orpellets. Tablets may also be multi-layered. Multi-layered tablets areespecially preferred when the active ingredients have markedly differentpharmacokinetic profiles. Optionally, the finished tablet may be coatedor uncoated.

The coating composition may comprise an insoluble matrix polymer and/ora water soluble material. Water soluble materials can be polymers suchas polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, or monomericmaterials such as sugars (e.g., lactose, sucrose, fructose, mannitol andthe like), salts (e.g., sodium chloride, potassium chloride and thelike), organic acids (e.g., fumaric acid, succinic acid, lactic acid,and tartaric acid), and mixtures thereof. Optionally, an enteric polymermay be incorporated into the coating composition. Suitable entericpolymers include hydroxypropyl methyl cellulose, acetate succinate,hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate phthalate,cellulose acetate phthalate, cellulose acetate trimellitate, shellac,zein, and polymethacrylates containing carboxyl groups. The coatingcomposition may be plasticised by adding suitable plasticisers such as,for example, diethyl phthalate, citrate esters, polyethylene glycol,glycerol, acetylated glycerides, acetylated citrate esters,dibutylsebacate, and castor oil. The coating composition may alsoinclude a filler, which can be an insoluble material such as silicondioxide, titanium dioxide, talc, kaolin, alumina, starch, powderedcellulose, MCC, or polacrilin potassium. The coating composition may beapplied as a solution or latex in organic solvents or aqueous solventsor mixtures thereof. Solvents such as water, lower alcohol, lowerchlorinated hydrocarbons, ketones, or mixtures thereof may be used.

The compositions of the invention may be formulated using a variety ofexcipients. Suitable excipients include microcrystalline cellulose (e.g.Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate,methyl methacrylate, trimethylammonioethyl methacrylate chloride) (suchas Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M,Premium CR Methocel K100M, Methocel E5, Opadry®), magnesium stearate,talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®),and protamine sulfate. The slow release agent may also comprise acarrier, which can comprise, for example, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents. Pharmaceutically acceptable salts can also be used inthese slow release agents, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates, or sulfates, as well as thesalts of organic acids such as acetates, proprionates, malonates, orbenzoates. The composition may also contain liquids, such as water,saline, glycerol, and ethanol, as well as substances such as wettingagents, emulsifying agents, or pH buffering agents. Liposomes may alsobe used as a carrier.

In another embodiment, the compositions of the present invention areencapsulated in liposomes, which have demonstrated utility in deliveringbeneficial active agents in a controlled manner over prolonged periodsof time. Liposomes are closed bilayer membranes containing an entrappedaqueous volume. Liposomes may also be unilamellar vesicles possessing asingle membrane bilayer or multilamellar vesicles with multiple membranebilayers, each separated from the next by an aqueous layer. Thestructure of the resulting membrane bilayer is such that the hydrophobic(non-polar) tails of the lipid are oriented toward the center of thebilayer while the hydrophilic (polar) heads orient towards the aqueousphase. In one embodiment, the liposome may be coated with a flexiblewater soluble polymer that avoids uptake by the organs of themononuclear phagocyte system, primarily the liver and spleen. Suitablehydrophilic polymers for surrounding the liposomes include, withoutlimitation, PEG, polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences asdescribed in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094,the contents of which are incorporated by reference in their entirety.

Liposomes may be comprised of any lipid or lipid combination known inthe art. For example, the vesicle-forming lipids may benaturally-occurring or synthetic lipids, including phospholipids, suchas phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid,phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, andsphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104.The vesicle-forming lipids may also be glycolipids, cerebrosides, orcationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane(DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3[N—(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-ChoI); ordimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No.6,056,973. Cholesterol may also be present in the proper range to impartstability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and5,874,104.

Additional liposomal technologies are described in U.S. Pat. Nos.6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966;6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and4,684,479, the contents of which are incorporated herein by reference.These describe liposomes and lipid-coated microbubbles, and methods fortheir manufacture. Thus, one skilled in the art, considering both thedisclosure of this invention and the disclosures of these other patentscould produce a liposome for the extended release of the polypeptides ofthe present invention.

For liquid formulations, a desired property is that the formulation besupplied in a form that can pass through a 25, 28, 30, 31, 32 gaugeneedle for intravenous, intramuscular, intraarticular, or subcutaneousadministration.

Administration via transdermal formulations can be performed usingmethods also known in the art, including those described generally in,e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770, 4,861,800, 6,743,211,6,945,952, 4,284,444, and WO 89/09051, incorporated herein by referencein their entireties. A transdermal patch is a particularly usefulembodiment with polypeptides having absorption problems. Patches can bemade to control the release of skin-permeable active ingredients over a12 hour, 24 hour, 3 day, and 7 day period. In one example, a 2-folddaily excess of a polypeptide of the present invention is placed in anon-volatile fluid. The compositions of the invention are provided inthe form of a viscous, non-volatile liquid. The penetration through skinof specific formulations may be measures by standard methods in the art(for example, Franz et al., J. Invest. Derm. 64:194-195 (1975)).Examples of suitable patches are passive transfer skin patches,iontophoretic skin patches, or patches with microneedles such asNicoderm.

In other embodiments, the composition may be delivered via intranasal,buccal, or sublingual routes to the brain to enable transfer of theactive agents through the olfactory passages into the CNS and reducingthe systemic administration. Devices commonly used for this route ofadministration are included in U.S. Pat. No. 6,715,485. Compositionsdelivered via this route may enable increased CNS dosing or reducedtotal body burden reducing systemic toxicity risks associated withcertain drugs. Preparation of a pharmaceutical composition for deliveryin a subdermally implantable device can be performed using methods knownin the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518;5,660,848; and 5,756,115.

Osmotic pumps may be used as slow release agents in the form of tablets,pills, capsules or implantable devices. Osmotic pumps are well known inthe art and readily available to one of ordinary skill in the art fromcompanies experienced in providing osmotic pumps for extended releasedrug delivery. Examples are ALZA's DUROS™; ALZA's OROS™; OsmoticaPharmaceutical's Osmodex™ system; Shire Laboratories' EnSoTrol™ system;and Alzet™. Patents that describe osmotic pump technology are U.S. Pat.Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532;6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776;4,200,0984; and 4,088,864, the contents of which are incorporated hereinby reference. One skilled in the art, considering both the disclosure ofthis invention and the disclosures of these other patents could producean osmotic pump for the extended release of the polypeptides of thepresent invention.

Syringe pumps may also be used as slow release agents. Such devices aredescribed in U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370;6,254,573; 4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502;5,492,534; 5,318,540; and 4,988,337, the contents of which areincorporated herein by reference. One skilled in the art, consideringboth the disclosure of this invention and the disclosures of these otherpatents could produce a syringe pump for the extended release of thecompositions of the present invention.

IX). Pharmaceutical Kits

In another aspect, the invention provides a kit to facilitate the use ofthe CFXTEN polypeptides. The kit comprises the pharmaceuticalcomposition provided herein, a label identifying the pharmaceuticalcomposition, and an instruction for storage, reconstitution and/oradministration of the pharmaceutical compositions to a subject. In someembodiment, the kit comprises, preferably: (a) an amount of a CFXTENfusion protein composition sufficient to treat a disease, condition ordisorder upon administration to a subject in need thereof; and (b) anamount of a pharmaceutically acceptable carrier; together in aformulation ready for injection or for reconstitution with sterilewater, buffer, or dextrose; together with a label identifying the CFXTENdrug and storage and handling conditions, and a sheet of the approvedindications for the drug, instructions for the reconstitution and/oradministration of the CFXTEN drug for the use for the prevention and/ortreatment of a approved indication, appropriate dosage and safetyinformation, and information identifying the lot and expiration of thedrug. In another embodiment of the foregoing, the kit can comprise asecond container that can carry a suitable diluent for the CFXTENcomposition, the use of which will provide the user with the appropriateconcentration of CFXTEN to be delivered to the subject.

EXAMPLES Example 1: Construction of XTEN_AD36 Motif Segments

The following example describes the construction of a collection ofcodon-optimized genes encoding motif sequences of 36 amino acids. As afirst step, a stuffer vector pCW0359 was constructed based on a pETvector and that includes a T7 promoter. pCW0359 encodes a cellulosebinding domain (CBD) and a TEV protease recognition site followed by astuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaIand BbsI sites were inserted such that they generate compatibleoverhangs after digestion. The stuffer sequence is followed by atruncated version of the GFP gene and a His tag. The stuffer sequencecontains stop codons and thus E. coli cells carrying the stuffer plasmidpCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 wasdigested with BsaI and KpnI to remove the stuffer segment and theresulting vector fragment was isolated by agarose gel purification. Thesequences were designated XTEN_AD36, reflecting the AD family of motifs.Its segments have the amino acid sequence [X]₃ where X is a 12merpeptide with the sequences: GESPGGSSGSES (SEQ ID NO: 31), GSEGSSGPGESS(SEQ ID NO: 32), GSSESGSSEGGP (SEQ ID NO: 33), or GSGGEPSESGSS (SEQ IDNO: 34). The insert was obtained by annealing the following pairs ofphosphorylated synthetic oligonucleotide pairs:

AD1for: (SEQ ID NO: 132) AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1rev:(SEQ ID NO: 133) ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for:(SEQ ID NO: 134) AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev:(SEQ ID NO: 135) ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT AD3for:(SEQ ID NO: 136) AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev:(SEQ ID NO: 137) ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA AD4for:(SEQ ID NO: 138) AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0401 showed green fluorescence afterinduction, which shows that the sequence of XTEN_AD36 had been ligatedin frame with the GFP gene and that most sequences of XTEN_AD36 had goodexpression levels.

We screened 96 isolates from library LCW0401 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 39 clones were identified thatcontained correct XTEN_AD36 segments. The file names of the nucleotideand amino acid constructs for these segments are listed in Table 9.

TABLE 9 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQ ID IDFile name Amino acid sequence NO: Nucleotide sequence NO:LCW0401_001_GFP-N_A01.ab1 GSGGEPSESGSSGESPGG 141GGTTCTGGTGGCGAACCGTCCGAGTC 142 SSGSESGESPGGSSGSESTGGTAGCTCAGGTGAATCTCCGGGTG GCTCTAGCGGTTCCGAGTCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCCGA GTCA LCW0401_002_GFP-N_B01.ab1GSEGSSGPGESSGESPGG 143 GGTAGCGAAGGTTCTTCTGGTCCTGG 144 SSGSESGSSESGSSEGGPCGAGTCTTCAGGTGAATCTCCTGGTG GTTCCAGCGGTTCTGAATCAGGTTCCTCCGAAAGCGGTTCTTCCGAGGGCG GTCCA LCW0401_003_GFP-N_C01.ab1GSSESGSSEGGPGSSESG 145 GGTTCCTCTGAAAGCGGTTCTTCCGA 146 SSEGGPGESPGGSSGSESAGGTGGTCCAGGTTCCTCTGAAAGCG GTTCTTCTGAGGGTGGTCCAGGTGAATCTCCGGGTGGCTCCAGCGGTTCCGA GTCA LCW0401_004_GFP-N_D01.ab1GSGGEPSESGSSGSSESG 147 GGTTCCGGTGGCGAACCGTCTGAATC 148 SSEGGPGSGGEPSESGSSTGGTAGCTCAGGTTCTTCTGAAAGCG GTTCTTCCGAGGGTGGTCCAGGTTCTGGTGGTGAACCTTCCGAGTCTGGTAG CTCA LCW0401_007_GFP-N_F01.ab1GSSESGSSEGGPGSEGSS 149 GGTTCTTCCGAAAGCGGTTCTTCTGA 150 GPGESSGSEGSSGPGESSGGGTGGTCCAGGTAGCGAAGGTTCTT CCGGTCCAGGTGAGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATC TTCA LCW0401_008_GFP-N_G01.ab1GSSESGSSEGGPGESPGG 151 GGTTCCTCTGAAAGCGGTTCTTCCGA 152 SSGSESGSEGSSGPGESSGGGTGGTCCAGGTGAATCTCCAGGT GGTTCCAGCGGTTCTGAGTCAGGTAGCGAAGGTTCTTCTGGTCCAGGTGAAT CCTCA LCW0401_012_GFP-N_H01.ab1GSGGEPSESGSSGSGGEP 153 GGTTCTGGTGGTGAACCGTCTGAGTC 154 SESGSSGSEGSSGPGESSTGGTAGCTCAGGTTCCGGTGGCGAAC CATCCGAATCTGGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTC TTCA LCW0401_015_GFP-N_A02.ab1GSSESGSSEGGPGSEGSS 155 GGTTCTTCCGAAAGCGGTTCTTCCGA 156 GPGESSGESPGGSSGSESAGGCGGTCCAGGTAGCGAAGGTTCT TCTGGTCCAGGCGAATCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCTG AGTCA LCW0401_016_GFP-N_B02.ab1GSSESGSSEGGPGSSESG 157 GGTTCCTCCGAAAGCGGTTCTTCTGA 158 SSEGGPGSSESGSSEGGPGGGCGGTCCAGGTTCCTCCGAAAGC GGTTCTTCCGAGGGCGGTCCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGCG GTCCA LCW0401_020_GFP-N_E02.ab1GSGGEPSESGSSGSEGSS 159 GGTTCCGGTGGCGAACCGTCCGAATC 160 GPGESSGSSESGSSEGGPTGGTAGCTCAGGTAGCGAAGGTTCTT CTGGTCCAGGCGAATCTTCAGGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGG TCCA LCW0401_022_GFP-N_F02.ab1GSGGEPSESGSSGSSESG 161 GGTTCTGGTGGTGAACCGTCCGAATC 162 SSEGGPGSGGEPSESGSSTGGTAGCTCAGGTTCTTCCGAAAGCG GTTCTTCTGAAGGTGGTCCAGGTTCCGGTGGCGAACCTTCTGAATCTGGTAG CTCA LCW0401_024_GFP-N_G02.ab1GSGGEPSESGSSGSSESG 163 GGTTCTGGTGGCGAACCGTCCGAATC 164 SSEGGPGESPGGSSGSESTGGTAGCTCAGGTTCCTCCGAAAGCG GTTCTTCTGAAGGTGGTCCAGGTGAATCTCCAGGTGGTTCTAGCGGTTCTGA ATCA LCW0401_026_GFP-N_H02.ab1GSGGEPSESGSSGESPGG 165 GGTTCTGGTGGCGAACCGTCTGAGTC 166 SSGSESGSEGSSGPGESSTGGTAGCTCAGGTGAATCTCCTGGTG GCTCCAGCGGTTCTGAATCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATC TTCA LCW0401_027_GFP-N_A03.ab1GSGGEPSESGSSGESPGG 167 GGTTCCGGTGGCGAACCTTCCGAATC 168 SSGSESGSGGEPSESGSSTGGTAGCTCAGGTGAATCTCCGGGTG GTTCTAGCGGTTCTGAGTCAGGTTCTGGTGGTGAACCTTCCGAGTCTGGTAG CTCA LCW0401_028_GFP-N_B03.ab1GSSESGSSEGGPGSSESG 169 GGTTCCTCTGAAAGCGGTTCTTCTGA 170 SSEGGPGSSESGSSEGGPGGGCGGTCCAGGTTCTTCCGAAAGC GGTTCTTCCGAGGGCGGTCCAGGTTCTTCCGAAAGCGGTTCTTCTGAAGGCG GTCCA LCW0401_030_GFP-N_C03.ab1GESPGGSSGSESGSEGSS 171 GGTGAATCTCCGGGTGGCTCCAGCG 172 GPGESSGSEGSSGPGESSGTTCTGAGTCAGGTAGCGAAGGTTCT TCCGGTCCGGGTGAGTCCTCAGGTAGCGAAGGTTCTTCCGGTCCTGGTGAGT CTTCA LCW0401_031_GFP-N_D03.ab1GSGGEPSESGSSGSGGEP 173 GGTTCTGGTGGCGAACCTTCCGAATC 174 SESGSSGSSESGSSEGGPTGGTAGCTCAGGTTCCGGTGGTGAAC CTTCTGAATCTGGTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGG TCCA LCW0401_033_GFP-N_E03.ab1GSGGEPSESGSSGSGGEP 175 GGTTCCGGTGGTGAACCTTCTGAATC 176 SESGSSGSGGEPSESGSSTGGTAGCTCAGGTTCCGGTGGCGAAC CATCCGAGTCTGGTAGCTCAGGTTCCGGTGGTGAACCATCCGAGTCTGGTA GCTCA LCW0401_037_GFP-N_F03.ab1GSGGEPSESGSSGSSESG 177 GGTTCCGGTGGCGAACCTTCTGAATC 178 SSEGGPGSEGSSGPGESSTGGTAGCTCAGGTTCCTCCGAAAGCG GTTCTTCTGAGGGCGGTCCAGGTAGCGAAGGTTCTTCTGGTCCGGGCGAGTC TTCA LCW0401_038_GFP-N_G03.ab1GSGGEPSESGSSGSEGSS 179 GGTTCCGGTGGTGAACCGTCCGAGTC 180 GPGESSGSGGEPSESGSSTGGTAGCTCAGGTAGCGAAGGTTCTT CTGGTCCGGGTGAGTCTTCAGGTTCTGGTGGCGAACCGTCCGAATCTGGTA GCTCA LCW0401_039_GFP-N_H03.ab1GSGGEPSESGSSGESPGG 181 GGTTCTGGTGGCGAACCGTCCGAATC 182 SSGSESGSGGEPSESGSSTGGTAGCTCAGGTGAATCTCCTGGTG GTTCCAGCGGTTCCGAGTCAGGTTCTGGTGGCGAACCTTCCGAATCTGGTAG CTCA LCW0401_040_GFP-N_A04.ab1GSSESGSSEGGPGSGGEP 183 GGTTCTTCCGAAAGCGGTTCTTCCGA 184 SESGSSGSSESGSSEGGPGGGCGGTCCAGGTTCCGGTGGTGAA CCATCTGAATCTGGTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCTGAAGGTG GTCCA LCW0401_042_GFP-N_C04.ab1GSEGSSGPGESSGESPGG 185 GGTAGCGAAGGTTCTTCCGGTCCTGG 186 SSGSESGSEGSSGPGESSTGAGTCTTCAGGTGAATCTCCAGGTG GCTCTAGCGGTTCCGAGTCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTC CTCA LCW0401_046_GFP-N_D04.ab1GSSESGSSEGGPGSSESG 187 GGTTCCTCTGAAAGCGGTTCTTCCGA 188 SSEGGPGSSESGSSEGGPAGGCGGTCCAGGTTCTTCCGAAAGC GGTTCTTCTGAGGGCGGTCCAGGTTCCTCCGAAAGCGGTTCTTCTGAGGGTG GTCCA LCW0401_047_GFP-N_E04.ab1GSGGEPSESGSSGESPGG 189 GGTTCTGGTGGCGAACCTTCCGAGTC 190 SSGSESGESPGGSSGSESTGGTAGCTCAGGTGAATCTCCGGGTG GTTCTAGCGGTTCCGAGTCAGGTGAATCTCCGGGTGGTTCCAGCGGTTCTGA GTCA LCW0401_051_GFP-N_F04.ab1GSGGEPSESGS SGSEGSS 191 GGTTCTGGTGGCGAACCATCTGAGTC 192GPGESSGESPGGSSGSES TGGTAGCTCAGGTAGCGAAGGTTCTT CCGGTCCAGGCGAGTCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCTG AGTCA LCW0401_053_GFP-N_H04.ab1GESPGGSSGSESGESPGG 193 GGTGAATCTCCTGGTGGTTCCAGCGG 194 SSGSESGESPGGSSGSESTTCCGAGTCAGGTGAATCTCCAGGTG GCTCTAGCGGTTCCGAGTCAGGTGAATCTCCTGGTGGTTCTAGCGGTTCTGA ATCA LCW0401_054_GFP-N_A05.ab1GSEGSSGPGESSGSEGSS 195 GGTAGCGAAGGTTCTTCCGGTCCAGG 196 GPGESSGSGGEPSESGSSTGAATCTTCAGGTAGCGAAGGTTCTT CTGGTCCTGGTGAATCCTCAGGTTCCGGTGGCGAACCATCTGAATCTGGTA GCTCA LCW0401_059_GFP-N_D05.ab1GSGGEPSESGSSGSEGSS 197 GGTTCTGGTGGCGAACCATCCGAATC 198 GPGESSGESPGGSSGSESTGGTAGCTCAGGTAGCGAAGGTTCTT CTGGTCCTGGCGAATCTTCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCCGA ATCA LCW0401_060_GFP-N_E05.ab1GSGGEPSESGSSGSSESG 199 GGTTCCGGTGGTGAACCGTCCGAATC 200 SSEGGPGSGGEPSESGSSTGGTAGCTCAGGTTCCTCTGAAAGCG GTTCTTCCGAGGGTGGTCCAGGTTCCGGTGGTGAACCTTCTGAGTCTGGTAG CTCA LCW0401_061_GFP-N_F05.ab1GSSESGSSEGGPGSGGEP 201 GGTTCCTCTGAAAGCGGTTCTTCTGA 202 SESGSSGSEGSSGPGESSGGGCGGTCCAGGTTCTGGTGGCGAA CCATCTGAATCTGGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCGGGTGAAT CTTCA LCW0401_063_GFP-N_H05.ab1GSGGEPSESGSSGSEGSS 203 GGTTCTGGTGGTGAACCGTCCGAATC 204 GPGESSGSEGSSGPGESSTGGTAGCTCAGGTAGCGAAGGTTCTT CTGGTCCTGGCGAGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATC TTCA LCW0401_066_GFP-N_B06.ab1GSGGEPSESGSSGSSESG 205 GGTTCTGGTGGCGAACCATCCGAGTC 206 SSEGGPGSGGEPSESGSSTGGTAGCTCAGGTTCTTCCGAAAGCG GTTCTTCCGAAGGCGGTCCAGGTTCTGGTGGTGAACCGTCCGAATCTGGTA GCTCA LCW0401_067_GFP-N_C06.ab1GSGGEPSESGSSGESPGG 207 GGTTCCGGTGGCGAACCTTCCGAATC 208 SSGSESGESPGGSSGSESTGGTAGCTCAGGTGAATCTCCGGGTG GTTCTAGCGGTTCCGAATCAGGTGAATCTCCAGGTGGTTCTAGCGGTTCCGA ATCA LCW0401_069_GFP-N_D06.ab1GSGGEPSESGSSGSGGEP 209 GGTTCCGGTGGTGAACCATCTGAGTC 210 SESGSSGESPGGSSGSESTGGTAGCTCAGGTTCCGGTGGCGAAC CGTCCGAGTCTGGTAGCTCAGGTGAATCTCCGGGTGGTTCCAGCGGTTCCGA ATCA LCW0401_070_GFP-N_E06.ab1GSEGSSGPGESSGSSESG 211 GGTAGCGAAGGTTCTTCTGGTCCGGG 212 SSEGGPGSEGSSGPGESSCGAATCCTCAGGTTCCTCCGAAAGCG GTTCTTCCGAAGGTGGTCCAGGTAGCGAAGGTTCTTCCGGTCCTGGTGAATC TTCA LCW0401_078_GFP-N_F06.ab1GSSESGSSEGGPGESPGG 213 GGTTCCTCTGAAAGCGGTTCTTCTGA 214 SSGSESGESPGGSSGSESAGGCGGTCCAGGTGAATCTCCGGGT GGCTCCAGCGGTTCTGAATCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCCG AGTCA LCW0401_079_GFP-N_G06.ab1GSEGSSGPGESSGSEGSS 215 GGTAGCGAAGGTTCTTCTGGTCCAGG 216 GPGESSGSGGEPSESGSSCGAGTCTTCAGGTAGCGAAGGTTCTT CCGGTCCTGGCGAGTCTTCAGGTTCCGGTGGCGAACCGTCCGAATCTGGTA GCTCA

Example 2: Construction of XTEN_AE36 Segments

A codon library encoding XTEN sequences of 36 amino acid length wasconstructed. The XTEN sequence was designated XTEN_AE36. Its segmentshave the amino acid sequence [X]₃ where X is a 12mer peptide with thesequence: GSPAGSPTSTEE (SEQ ID NO: 35), GSEPATSGSE TP (SEQ ID NO: 36),GTSESA TPESGP (SEQ ID NO: 37), or GTSTEPSEGSAP (SEQ ID NO: 38). Theinsert was obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

AE1for: (SEQ ID NO: 217) AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AE1rev:(SEQ ID NO: 218) ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AE2for:(SEQ ID NO: 219) AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC AE2rev:(SEQ ID NO: 220) ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3for:(SEQ ID NO: 221) AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev:(SEQ ID NO: 222) ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for:(SEQ ID NO: 223) AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4rev:(SEQ ID NO: 224) ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0402 showed green fluorescence afterinduction which shows that the sequence of XTEN_AE36 had been ligated inframe with the GFP gene and most sequences of XTEN_AE36 show goodexpression.

We screened 96 isolates from library LCW0402 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 37 clones were identified thatcontained correct XTEN_AE36 segments. The file names of the nucleotideand amino acid constructs for these segments are listed in Table 10.

TABLE 10 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQ ID IDFile name Amino acid sequence NO: Nucleotide sequence NO:LCW0402_002_GFP-N_A07.ab1 GSPAGSPTSTEEGTSE 225GGTAGCCCGGCAGGCTCTCCGACCTCTA 226 SATPESGPGTSTEPSECTGAGGAAGGTACTTCTGAAAGCGCAAC GSAP CCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA LCW0402_003_GFP-N_B07.ab1 GTSTEPSEGSAPGTST 227GGTACTTCTACCGAACCGTCCGAAGGCA 228 EPSEGSAPGTSTEPSEGCGCTCCAGGTACCTCTACTGAACCTTCC GSAP GAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA LCW0402_004_GFP-N_C07.ab1 GTSTEPSEGSAPGTSE 229GGTACCTCTACCGAACCGTCTGAAGGTA 230 SATPESGPGTSESATPGCGCACCAGGTACCTCTGAAAGCGCAAC ESGP TCCTGAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA LCW0402_005_GFP-N_D07.ab1 GTSTEPSEGSAPGTSE 231GGTACTTCTACTGAACCGTCTGAAGGTA 232 SATPESGPGTSESATPGCGCACCAGGTACTTCTGAAAGCGCAAC ESGP CCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCW0402_006_GFP-N_E07.ab1 GSEPATSGSETPGTSE 233GGTAGCGAACCGGCAACCTCCGGCTCTG 234 SATPESGPGSPAGSPTAAACCCCAGGTACCTCTGAAAGCGCTAC STEE TCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA LCW0402_008_GFP-N_F07.ab1 GTSESATPESGPGSEP 235GGTACTTCTGAAAGCGCAACCCCTGAAT 236 ATSGSETPGTSTEPSECCGGTCCAGGTAGCGAACCGGCTACTTC GSAP TGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA LCW0402_009_GFP-N_G07.ab1 GSPAGSPTSTEEGSPA 237GGTAGCCCGGCTGGCTCTCCAACCTCCA 238 GSPTSTEEGSEPATSGCTGAGGAAGGTAGCCCGGCTGGCTCTCC SETP AACCTCCACTGAAGAAGGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA LCW0402_011_GFP-N_A08.ab1 GSPAGSPTSTEEGTSE 239GGTAGCCCGGCTGGCTCTCCTACCTCTAC 240 SATPESGPGTSTEPSETGAGGAAGGTACTTCTGAAAGCGCTACT GSAP CCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA LCW0402_012_GFP-N_B08.ab1 GSPAGSPTSTEEGSPA 241GGTAGCCCTGCTGGCTCTCCGACTTCTAC 242 GSPTSTEEGTSTEPSETGAGGAAGGTAGCCCGGCTGGTTCTCCG GSAP ACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA LCW0402_013_GFP-N_C08.ab1 GTSESATPESGPGTST 243GGTACTTCTGAAAGCGCTACTCCGGAGT 244 EPSEGSAPGTSTEPSECCGGTCCAGGTACCTCTACCGAACCGTC GSAP CGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCW0402_014_GFP-N_D08.ab1 GTSTEPSEGSAPGSPA 245GGTACCTCTACCGAACCTTCCGAAGGTA 246 GSPTSTEEGTSTEPSEGCGCTCCAGGTAGCCCGGCAGGTTCTCC GSAP TACTTCCACTGAGGAAGGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA LCW0402_015_GFP-N_E08.ab1 GSEPATSGSETPGSPA 247GGTAGCGAACCGGCTACTTCCGGCTCTG 248 GSPTSTEEGTSESATPAGACTCCAGGTAGCCCTGCTGGCTCTCC ESGP GACCTCTACCGAAGAAGGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA LCW0402_016_GFP-N_F08.ab1 GTSTEPSEGSAPGTSE 249GGTACTTCTACCGAACCTTCCGAGGGCA 250 SATPESGPGTSESATPGCGCACCAGGTACTTCTGAAAGCGCTAC ESGP CCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA LCW0402_020_GFP-N_G08.ab1 GTSTEPSEGSAPGSEP 251GGTACTTCTACTGAACCGTCTGAAGGCA 252 ATSGSETPGSPAGSPTGCGCACCAGGTAGCGAACCGGCTACTTC STEE CGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA LCW0402_023_GFP-N_A09.ab1 GSPAGSPTSTEEGTSE 253GGTAGCCCTGCTGGCTCTCCAACCTCCAC 254 SATPESGPGSEPATSGCGAAGAAGGTACCTCTGAAAGCGCAACC SETP CCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA LCW0402_024_GFP-N_B09.ab1 GTSESATPESGPGSPA 255GGTACTTCTGAAAGCGCTACTCCTGAGTC 256 GSPTSTEEGSPAGSPTCGGCCCAGGTAGCCCGGCTGGCTCTCCG STEE ACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA LCW0402_025_GFP-N_C09.ab1 GTSTEPSEGSAPGTSE 257GGTACCTCTACTGAACCTTCTGAGGGCA 258 SATPESGPGTSTEPSEGCGCTCCAGGTACTTCTGAAAGCGCTAC GSAP CCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_026_GFP-N_D09.ab1 GSPAGSPTSTEEGTST 259GGTAGCCCGGCAGGCTCTCCGACTTCCA 260 EPSEGSAPGSEPATSGCCGAGGAAGGTACCTCTACTGAACCTTC SETP TGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA LCW0402_027_GFP-N_E09.ab1 GSPAGSPTSTEEGTST 261GGTAGCCCAGCAGGCTCTCCGACTTCCA 262 EPSEGSAPGTSTEPSECTGAGGAAGGTACTTCTACTGAACCTTCC GSAP GAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA LCW0402_032_GFP-N_H09.ab1 GSEPATSGSETPGTSE 263GGTAGCGAACCTGCTACCTCCGGTTCTG 264 SATPESGPGSPAGSPTAAACCCCAGGTACCTCTGAAAGCGCAAC STEE TCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA LCW0402_034_GFP-N_A10.ab1 GTSESATPESGPGTST 265GGTACCTCTGAAAGCGCTACTCCGGAGT 266 EPSEGSAPGTSTEPSECTGGCCCAGGTACCTCTACTGAACCGTCT GSAP GAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_036_GFP-N_C10.ab1 GSPAGSPTSTEEGTST 267GGTAGCCCGGCTGGTTCTCCGACTTCCAC 268 EPSEGSAPGTSTEPSECGAGGAAGGTACCTCTACTGAACCTTCT GSAP GAGGGTAGCGCTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA LCW0402_039_GFP-N_E10.ab1 GTSTEPSEGSAPGTST 269GGTACTTCTACCGAACCGTCCGAGGGCA 270 EPSEGSAPGTSTEPSEGCGCTCCAGGTACTTCTACTGAACCTTCT GSAP GAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW0402_040_GFP-N_F10.ab1 GSEPATSGSETPGTSE 271GGTAGCGAACCTGCAACCTCTGGCTCTG 272 SATPESGPGTS1EPSEAAACCCCAGGTACCTCTGAAAGCGCTAC GSAP TCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA LCW0402_041_GFP-N_G10.ab1 GTSTEPSEGSAPGSPA 273GGTACTTCTACCGAACCGTCCGAGGGTA 274 GSPTSTEEGTSTEPSEGCGCACCAGGTAGCCCAGCAGGTTCTCC GSAP TACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA LCW0402_050_GFP-N_A11.ab1 GSEPATSGSETPGTSE 275GGTAGCGAACCGGCAACCTCCGGCTCTG 276 SATPESGPGSEPATSGAAACTCCAGGTACTTCTGAAAGCGCTAC SETP TCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA LCW0402_051_GFP-N_B11.ab1 GSEPATSGSETPGTSE 277GGTAGCGAACCGGCAACTTCCGGCTCTG 278 SATPESGPGSEPATSGAAACCCCAGGTACTTCTGAAAGCGCTAC SETP TCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA LCW0402_059_GFP-N_E11.ab1 GSEPATSGSETPGSEP 279GGTAGCGAACCGGCAACCTCTGGCTCTG 280 ATSGSETPGTSTEPSEAAACTCCAGGTAGCGAACCTGCAACCTC GSAP CGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA LCW0402_060_GFP-N_F11.ab1 GTSESATPESGPGSEP 281GGTACTTCTGAAAGCGCTACCCCGGAAT 282 ATSGSETPGSEPATSGCTGGCCCAGGTAGCGAACCGGCTACTTC SETP TGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA LCW0402_061_GFP-N_G11.ab1 GTSTEPSEGSAPGTST 283GGTACCTCTACTGAACCTTCCGAAGGCA 284 EPSEGSAPGTSESATPGCGCTCCAGGTACCTCTACCGAACCGTC ESGP CGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA LCW0402_065_GFP-N_A12.ab1 GSEPATSGSETPGTSE 285GGTAGCGAACCGGCAACCTCTGGCTCTG 286 SATPESGPGTSESATPAAACCCCAGGTACCTCTGAAAGCGCTAC ESGP TCCGGAATCTGGTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA LCW0402_066_GFP-N_B12.ab1 GSEPATSGSETPGSEP 287GGTAGCGAACCTGCTACCTCCGGCTCTG 288 ATSGSETPGTSTEPSEAAACTCCAGGTAGCGAACCGGCTACTTC GSAP CGGTTCTGAAACTCCAGGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA LCW0402_067_GFP-N_C12.ab1 GSEPATSGSETPGTST 289GGTAGCGAACCTGCTACTTCTGGTTCTGA 290 EPSEGSAPGSEPATSGAACTCCAGGTACTTCTACCGAACCGTCC SETP GAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW0402_069_GFP-N_D12.ab1 GTSTEPSEGSAPGTST 291GGTACCTCTACCGAACCGTCCGAGGGTA 292 EPSEGSAPGSEPATSGGCGCACCAGGTACCTCTACTGAACCGTC SETP TGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA LCW0402_073_GFP-N_F12.ab1 GTSTEPSEGSAPGSEP 293GGTACTTCTACTGAACCTTCCGAAGGTA 294 ATSGSETPGSPAGSPTGCGCTCCAGGTAGCGAACCTGCTACTTCT STEE GGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW0402_074_GFP-N_G12.ab1 GSEPATSGSETPGSPA 295GGTAGCGAACCGGCTACTTCCGGCTCTG 296 GSPTSTEEGTSESATPAGACTCCAGGTAGCCCAGCTGGTTCTCC ESGP AACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA LCW0402_075_GFP-N_H12.ab1 GTSESATPESGPGSEP 297GGTACCTCTGAAAGCGCAACTCCTGAGT 298 ATSGSETPGTSESATPCTGGCCCAGGTAGCGAACCTGCTACCTC ESGP CGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA

Example 3: Construction of XTEN_AF36 Segments

A codon library encoding sequences of 36 amino acid length wasconstructed. The sequences were designated XTEN_AF36. Its segments havethe amino acid sequence [X]3 where X is a 12mer peptide with thesequence: GSTSESPSGTAP (SEQ ID NO: 39), GTSTPESGSASP (SEQ ID NO: 40),GTSPSGESSTAP (SEQ ID NO: 41), or GSTSSTAESPGP (SEQ ID NO: 42). Theinsert was obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

AF1for: (SEQ ID NO: 299) AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC AF1rev:(SEQ ID NO: 300) ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA AF2for:(SEQ ID NO: 301) AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC AF2rev:(SEQ ID NO: 302) ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT AF3for:(SEQ ID NO: 303) AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC AF3rev:(SEQ ID NO: 304) ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT AF4for:(SEQ ID NO: 305) AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC AF4rev:(SEQ ID NO: 306) ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0403 showed green fluorescence afterinduction which shows that the sequence of XTEN_AF36 had been ligated inframe with the GFP gene and most sequences of XTEN_AF36 show goodexpression.

We screened 96 isolates from library LCW0403 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 44 clones were identified thatcontained correct XTEN_AF36 segments. The file names of the nucleotideand amino acid constructs for these segments are listed in Table 11.

TABLE 11 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQ ID IDFile name Amino acid sequence NO: Nucleotide sequence NO:LCW0403_004_GFP-N_A01.ab1 GTSTPESGSASPGTSP 307GGTACTTCTACTCCGGAAAGCGGTTCCG 308 SGESSTAPGTSPSGESCATCTCCAGGTACTTCTCCTAGCGGTGA STAP ATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_GFP-N_B01.ab1 GTSPSGESSTAPGSTS 309GGTACTTCTCCGAGCGGTGAATCTTCTA 310 STAESPGPGTSPSGESCCGCACCAGGTTCTACTAGCTCTACCGC STAP TGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_GFP-N_C01.ab1 GSTSSTAESPGPGTSP 311GGTTCCACCAGCTCTACTGCTGAATCTC 312 SGESSTAPGTSTPESGCTGGTCCAGGTACCTCTCCTAGCGGTGA SASP ATCTTCTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_GFP-N_D01.ab1 GSTSSTAESPGPGSTS 313GGTTCTACCAGCTCTACTGCAGAATCTC 314 STAESPGPGTSPSGESCTGGCCCAGGTTCCACCAGCTCTACCGC STAP AGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_GFP-N_E01.ab1 GSTSSTAESPGPGTSP 315GGTTCTACTAGCTCTACTGCTGAATCTCC 316 SGESSTAPGTSTPESGTGGCCCAGGTACTTCTCCTAGCGGTGAA SASP TCTTCTACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA LCW0403_010_GFP-N_F01.ab1 GSTSSTAESPGPGTST 317GGTTCTACCAGCTCTACCGCAGAATCTC 318 PESGSASPGSTSESPSCTGGTCCAGGTACCTCTACTCCGGAAAG GTAP CGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_011_GFP-N_G01.ab1 GSTSSTAESPGPGTST 319GGTTCTACTAGCTCTACTGCAGAATCTC 320 PESGSASPGTSTPESGCTGGCCCAGGTACCTCTACTCCGGAAAG SASP CGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_GFP-N_H01.ab1 GSTSESPSGTAPGTSP 321GGTTCTACCAGCGAATCTCCTTCTGGCA 322 SGESSTAPGSTSESPSCCGCTCCAGGTACCTCTCCTAGCGGCGA GTAP ATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_GFP-N_A02.ab1 GSTSSTAESPGPGSTS 323GGTTCCACCAGCTCTACTGCAGAATCTC 324 STAESPGPGTSPSGESCGGGCCCAGGTTCTACTAGCTCTACTGC STAP AGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_GFP-N_B02.ab1 GSTSSTAESPGPGTST 325GGTTCCACTAGCTCTACTGCAGAATCTC 326 PESGSASPGSTSESPSCTGGCCCAGGTACCTCTACCCCTGAAAG GTAP CGGCTCTGCATCTCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_GFP-N_C02.ab1 GSTSSTAESPGPGSTS 327GGTTCTACTAGCTCTACTGCTGAATCTCC 328 STAESPGPGTSPSGESGGGTCCAGGTTCTACCAGCTCTACTGCT STAP GAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA LCW0403_017_GFP-N_D02.ab1 GSTSSTAESPGPGSTS 329GGTTCTACCAGCTCTACCGCTGAATCTC 330 ESPSGTAPGSTSSTAECTGGCCCAGGTTCTACCAGCGAATCCCC SPGP GTCTGGCACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_GFP-N_E02.ab1 GSTSSTAESPGPGSTS 331GGTTCTACCAGCTCTACCGCAGAATCTC 332 STAESPGPGSTSSTAECTGGCCCAGGTTCCACTAGCTCTACCGC SPGP TGAATCTCCTGGTCCAGGTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCA LCW0403_019_GFP-N_F02.ab1 GSTSESPSGTAPGSTS 333GGTTCTACTAGCGAATCCCCTTCTGGTA 334 STAESPGPGSTSSTAECTGCTCCAGGTTCCACTAGCTCTACCGC SPGP TGAATCTCCTGGCCCAGGTTCCACTAGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_GFP-N_H02.ab1 GSTSESPSGTAPGSTS 335GGTTCTACTAGCGAATCTCCTTCTGGTA 336 ESPSGTAPGSTSESPSCCGCTCCAGGTTCTACCAGCGAATCCCC GTAP GTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCA LCW0403_024_GFP-N_A03.ab1 GSTSSTAESPGPGSTS 337GGTTCCACCAGCTCTACTGCTGAATCTC 338 STAESPGPGSTSSTAECTGGCCCAGGTTCTACCAGCTCTACTGC SPGP TGAATCTCCGGGCCCAGGTTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_GFP-N_B03.ab1 GSTSSTAESPGPGSTS 339GGTTCCACTAGCTCTACCGCAGAATCTC 340 STAESPGPGTSPSGESCTGGTCCAGGTTCTACTAGCTCTACTGCT STAP GAATCTCCGGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_GFP-N_D03.ab1 GSSPSASTGTGPGSST 341GGTTCTAGCCCTTCTGCTTCCACCGGTAC 342 PSGATGSPGSSTPSGACGGCCCAGGTAGCTCTACTCCGTCTGGT TGSP GCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA LCW0403_029_GFP-N_E03.ab1 GTSPSGESSTAPGTST 343GGTACTTCCCCTAGCGGTGAATCTTCTA 344 PESGSASPGSTSSTAECTGCTCCAGGTACCTCTACTCCGGAAAG SPGP CGGCTCCGCATCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_GFP-N_F03.ab1 GSTSSTAESPGPGSTS 345GGTTCTACTAGCTCTACCGCTGAATCTC 346 STAESPGPGTSTPESGCGGGTCCAGGTTCTACCAGCTCTACTGC SASP AGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_031_GFP-N_G03.ab1 GTSPSGESSTAPGSTS 347GGTACTTCTCCTAGCGGTGAATCTTCTA 348 STAESPGPGTSTPESGCCGCTCCAGGTTCTACCAGCTCTACTGC SASP TGAATCTCCTGGCCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA LCW0403_033_GFP-N_H03.ab1 GSTSESPSGTAPGSTS 349GGTTCTACTAGCGAATCCCCTTCTGGTA 350 STAESPGPGSTSSTAECTGCACCAGGTTCTACCAGCTCTACTGC SPGP TGAATCTCCGGGCCCAGGTTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_GFP-N_A04.ab1 GSTSSTAESPGPGSTS 351GGTTCCACCAGCTCTACCGCTGAATCTC 352 ESPSGTAPGSTSSTAECGGGCCCAGGTTCTACCAGCGAATCCCC SPGP TTCTGGCACTGCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_GFP-N_B04.ab1 GSTSSTAESPGPGTSP 353GGTTCTACCAGCTCTACTGCTGAATCTC 354 SGESSTAPGTSTPESGCGGGTCCAGGTACTTCCCCGAGCGGTGA SASP ATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_GFP-N_C04.ab1 GSTSESPSGTAPGSTS 355GGTTCTACCAGCGAATCTCCTTCTGGCA 356 ESPSGTAPGTSPSGESCCGCTCCAGGTTCTACTAGCGAATCCCC STAP GTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_GFP-N_D04.ab1 GSTSESPSGTAPGSTS 357GGTTCTACCAGCGAATCCCCTTCTGGTA 358 ESPSGTAPGTSTPESGCTGCTCCAGGTTCTACCAGCGAATCCCC SASP TTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_GFP-N_E04.ab1 GTSTPESGSASPGSTS 359GGTACCTCTACTCCTGAAAGCGGTTCTG 360 STAESPGPGSTSSTAECATCTCCAGGTTCCACTAGCTCTACCGC SPGP AGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_GFP-N_F04.ab1 GSTSESPSGTAPGSTS 361GGTTCTACCAGCGAATCCCCTTCTGGCA 362 ESPSGTAPGTSPSGESCTGCACCAGGTTCTACTAGCGAATCCCC STAP TTCTGGTACCGCACCAGGTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_GFP-N_G04.ab1 GSTSSTAESPGPGSTS 363GGTTCTACTAGCTCTACCGCTGAATCTC 364 STAESPGPGSTSESPSCTGGCCCAGGTTCCACTAGCTCTACCGC GTAP AGAATCTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_GFP-N_H04.ab1 GSTSSTAESPGPGSTS 365GGTTCCACCAGCTCTACTGCAGAATCTC 366 STAESPGPGTSTPESGCTGGCCCAGGTTCTACTAGCTCTACCGC SASP AGAATCTCCTGGTCCAGGTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_GFP-N_A05.ab1 GSTSSTAESPGPGSTS 367GGTTCTACTAGCTCTACTGCTGAATCTCC 368 STAESPGPGSTSESPSGGGCCCAGGTTCTACTAGCTCTACCGCT GTAP GAATCTCCGGGTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_GFP-N_B05.ab1 GTSPSGESSTAPGSTS 369GGTACCTCCCCGAGCGGTGAATCTTCTA 370 ESPSGTAPGSTSSTAECTGCACCAGGTTCTACTAGCGAATCCCC SPGP TTCTGGTACTGCTCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_GFP-N_C05.ab1 GSTSESPSGTAPGTSP 371GGTTCTACTAGCGAATCCCCGTCTGGTA 372 SGESSTAPGSTSSTAECTGCTCCAGGTACTTCCCCTAGCGGTGA SPGP ATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA LCW0403_057_GFP-N_D05.ab1 GSTSSTAESPGPGSTS 373GGTTCTACCAGCTCTACCGCTGAATCTC 374 ESPSGTAPGTSPSGESCTGGCCCAGGTTCTACTAGCGAATCTCC STAP GTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_GFP-N_E05.ab1 GSTSESPSGTAPGSTS 375GGTTCTACTAGCGAATCTCCTTCTGGCA 376 ESPSGTAPGTSTPESGCTGCACCAGGTTCTACCAGCGAATCTCC SASP GTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_GFP-N_F05.ab1 GTSTPESGSASPGSTS 377GGTACCTCTACTCCGGAAAGCGGTTCCG 378 ESPSGTAPGSTSSTAECATCTCCAGGTTCTACCAGCGAATCCCC SPGP GTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_GFP-N_G05.ab1 GSTSSTAESPGPGTSP 379GGTTCTACTAGCTCTACTGCAGAATCTC 380 SGESSTAPGTSPSGESCGGGCCCAGGTACCTCTCCTAGCGGTGA STAP ATCTTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_GFP-N_H05.ab1 GTSPSGESSTAPGTSP 381GGTACCTCCCCTAGCGGCGAATCTTCTA 382 SGESSTAPGTSPSGESCTGCTCCAGGTACCTCTCCTAGCGGCGA STAP ATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_GFP-N_A06.ab1 GSTSSTAESPGPGTST 383GGTTCCACTAGCTCTACTGCTGAATCTC 384 PESGSASPGSTSESPSCTGGCCCAGGTACTTCTACTCCGGAAAG GTAP CGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_GFP-N_B06.ab1 GSTSESPSGTAPGTSP 385GGTTCTACTAGCGAATCTCCGTCTGGCA 386 SGESSTAPGTSPSGESCTGCTCCAGGTACTTCTCCTAGCGGTGA STAP ATCTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_GFP-N_C06.ab1 GSTSESPSGTAPGTST 387GGTTCTACTAGCGAATCTCCTTCTGGTA 388 PESGSASPGSTSSTAECCGCTCCAGGTACTTCTACCCCTGAAAG SPGP CGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_068_GFP-N_D06.ab1 GSTSSTAESPGPGSTS 389GGTTCCACTAGCTCTACTGCTGAATCTC 390 STAESPGPGSTSESPSCTGGCCCAGGTTCTACCAGCTCTACCGC GTAP TGAATCTCCTGGCCCAGGTTCTACCAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_GFP-N_E06.ab1 GSTSESPSGTAPGTST 391GGTTCTACTAGCGAATCCCCGTCTGGTA 392 PESGSASPGTSTPESGCCGCACCAGGTACTTCTACCCCGGAAAG SASP CGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_GFP-N_F06.ab1 GSTSESPSGTAPGTST 393GGTTCTACTAGCGAATCCCCGTCTGGTA 394 PESGSASPGTSTPESGCTGCTCCAGGTACTTCTACTCCTGAAAG SASP CGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA

Example 4: Construction of XTEN_AG36 Segments

A codon library encoding sequences of 36 amino acid length wasconstructed. The sequences were designated XTEN_AG36. Its segments havethe amino acid sequence [X]₃ where X is a 12mer peptide with thesequence: GTPGSGTASSSP (SEQ ID NO: 43), GSSTPSGATGSP (SEQ ID NO: 44),GSSPSASTGTGP (SEQ ID NO: 45), or GASPGTSSTGSP (SEQ ID NO: 46). Theinsert was obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

AG1for: (SEQ ID NO: 395) AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC AG1rev:(SEQ ID NO: 396) ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT AG2for:(SEQ ID NO: 397) AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC AG2rev:(SEQ ID NO: 398) ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT AG3for:(SEQ ID NO: 399) AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC AG3rev:(SEQ ID NO: 400) ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA AG4for:(SEQ ID NO: 401) AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC AG4rev:(SEQ ID NO: 402) ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0404 showed green fluorescence afterinduction which shows that the sequence of XTEN_AG36 had been ligated inframe with the GFP gene and most sequences of XTEN_AG36 show goodexpression.

We screened 96 isolates from library LCW0404 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 44 clones were identified thatcontained correct XTEN_AG36 segments. The file names of the nucleotideand amino acid constructs for these segments are listed in Table 12.

TABLE 12 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQ ID IDFile name Amino acid sequence NO: Nucleotide sequence NO:LCW0404_001_GFP-N_A07.ab1 GASPGTSSTGSPGTPG 403GGTGCATCCCCGGGCACTAGCTCTACCG 404 SGTASSSPGSSTPSGAGTTCTCCAGGTACTCCTGGTAGCGGTAC TGSP TGCTTCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_GFP-N_B07.ab1 GSSTPSGATGSPGSSP 405GGTAGCTCTACCCCTTCTGGTGCTACCG 406 SASTGTGPGSSTPSGAGCTCTCCAGGTTCTAGCCCGTCTGCTTC TGSP TACCGGTACCGGTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_GFP-N_C07.ab1 GASPGTSSTGSPGSSP 407GGTGCATCTCCGGGTACTAGCTCTACCG 408 SASTGTGPGSSTPSGAGTTCTCCAGGTTCTAGCCCTTCTGCTTCC TGSP ACTGGTACCGGCCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCA LCW0404_007_GFP-N_D07.ab1 GTPGSGTASSSPGSST 409GGTACTCCGGGCAGCGGTACTGCTTCTT 410 PSGATGSPGASPGTSSCCTCTCCAGGTAGCTCTACCCCTTCTGG TGSP TGCAACTGGTTCCCCAGGTGCATCCCCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_GFP-N_E07.ab1 GTPGSGTASSSPGASP 411GGTACCCCTGGCAGCGGTACTGCTTCTT 412 GTSSTGSPGSRPSASTCTTCTCCAGGTGCTTCCCCTGGTACCAG GTGP CTCTACCGGTTCTCCAGGTTCTAGACCTTCTGCATCCACCGGTACTGGTCCA LCW0404_011_GFP-N_F07.ab1 GASPGTSSTGSPGSST 413GGTGCATCTCCTGGTACCAGCTCTACCG 414 PSGATGSPGASPGTSSGTTCTCCAGGTAGCTCTACTCCTTCTGG TGSP TGCTACTGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_012_GFP-N_G07.ab1 GTPGSGTASSSPGSST 415GGTACCCCGGGCAGCGGTACCGCATCTT 416 PSGATGSPGSSTPSGACCTCTCCAGGTAGCTCTACCCCGTCTGG TGSP TGCTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA LCW0404_014_GFP-N_H07.ab1 GASPGTSSTGSPGASP 417GGTGCATCTCCGGGCACTAGCTCTACTG 418 GTSSTGSPGASPGTSSGTTCTCCAGGTGCATCCCCTGGCACTAG TGSP CTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_GFP-N_A08.ab1 GSSTPSGATGSPGSSP 419GGTAGCTCTACTCCGTCTGGTGCAACCG 420 SASTGTGPGASPGTSSGCTCCCCAGGTTCTAGCCCGTCTGCTTC TGSP CACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_GFP-N_B08.ab1 GSSTPSGATGSPGSST 421GGTAGCTCTACTCCTTCTGGTGCTACCG 422 PSGATGSPGTPGSGTGTTCCCCAGGTAGCTCTACTCCTTCTGG ASSSP TGCTACTGGTTCCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_GFP-N_C08.ab1 GSSTPSGATGSPGSST 423GGTAGCTCTACTCCGTCTGGTGCAACCG 424 PSGATGSPGASPGTSSGTTCCCCAGGTAGCTCTACTCCTTCTGG TGSP TGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_018_GFP-N_D08.ab1 GTPGSGTASS SPGSSP 425GGTACTCCTGGTAGCGGTACCGCATCTT 426 SASTGTGPGSSTPSGACCTCTCCAGGTTCTAGCCCTTCTGCATCT TGSP ACCGGTACCGGTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCA LCW0404_023_GFP-N_F08.ab1 GASPGTSSTG SPGSSP 427GGTGCTTCCCCGGGCACTAGCTCTACCG 428 SASTGTGPGTPGSGTGTTCTCCAGGTTCTAGCCCTTCTGCATCT ASSSP ACTGGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_GFP-N_G08.ab1 GSSTPSGATGSPGSST 429GGTAGCTCTACTCCGTCTGGTGCTACCG 430 PSGATGSPGASPGTSSGCTCTCCAGGTAGCTCTACCCCTTCTGG TGSP TGCAACCGGCTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_GFP-N_A09.ab1 GTPGSGTASSSPGSST 431GGTACCCCTGGCAGCGGTACCGCTTCTT 432 PSGATGSPGSSPSASTCCTCTCCAGGTAGCTCTACCCCGTCTGG GTGP TGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCA LCW0404_030_GFP-N_B09.ab1 GSSTPSGATGSPGTPG 433GGTAGCTCTACTCCTTCTGGTGCAACCG 434 SGTASSSPGTPGSGTAGCTCCCCAGGTACCCCGGGCAGCGGTA SSSP CCGCATCTTCCTCTCCAGGTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCA LCW0404_031_GFP-N_C09.ab1 GTPGSGTASSSPGSST 435GGTACCCCGGGTAGCGGTACTGCTTCTT 436 PSGATGSPGASPGTSSCCTCTCCAGGTAGCTCTACCCCTTCTGG TGSP TGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA LCW0404_034_GFP-N_D09.ab1 GSSTPSGATGSPGSST 437GGTAGCTCTACCCCGTCTGGTGCTACCG 438 PSGATGSPGASPGTSSGCTCTCCAGGTAGCTCTACCCCGTCTGG TGSP TGCAACCGGCTCCCCAGGTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_GFP-N_E09.ab1 GASPGTSSTGSPGTPG 439GGTGCTTCTCCGGGCACCAGCTCTACTG 440 SGTASSSPGSSTPSGAGTTCTCCAGGTACCCCGGGCAGCGGTAC TGSP CGCATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCA LCW0404_036_GFP-N_F09.ab1 GSSPSASTGTGPGSST 441GGTTCTAGCCCGTCTGCTTCCACCGGTA 442 PSGATGSPGTPGSGTCTGGCCCAGGTAGCTCTACCCCGTCTGG ASSSP TGCAACTGGTTCCCCAGGTACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_GFP-N_G09.ab1 GASPGTSSTG SPGSSP 443GGTGCTTCTCCGGGCACCAGCTCTACTG 444 SASTGTGPGSSTPSGAGTTCTCCAGGTTCTAGCCCTTCTGCATC TGSP CACCGGTACCGGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_GFP-N_H09.ab1 GASPGTSSTGSPGSST 445GGTGCATCCCCGGGCACCAGCTCTACCG 446 PSGATGSPGSSTPSGAGTTCTCCAGGTAGCTCTACCCCGTCTGG TGSP TGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_GFP-N_A10.ab1 GTPGSGTASSSPGSST 447GGTACCCCTGGTAGCGGTACTGCTTCTT 448 PSGATGSPGTPGSGTCCTCTCCAGGTAGCTCTACTCCGTCTGG ASSSP TGCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_GFP-N_C10.ab1 GSSPSASTGTGPGSST 449GGTTCTAGCCCTTCTGCTTCCACCGGTA 450 PSGATGSPGSSTPSGACTGGCCCAGGTAGCTCTACCCCTTCTGG TGSP TGCTACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_GFP-N_D10.ab1 GASPGTSSTGSPGSSP 451GGTGCTTCTCCTGGCACCAGCTCTACTG 452 SASTGTGPGSSPSASTGTTCTCCAGGTTCTAGCCCTTCTGCTTCT GTGP ACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA LCW0404_047_GFP-N_F10.ab1 GTPGSGTASSSPGASP 453GGTACTCCTGGCAGCGGTACCGCTTCTT 454 GTSSTGSPGASPGTSSCTTCTCCAGGTGCTTCTCCTGGTACTAG TGSP CTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA LCW0404_048_GFP-N_G10.ab1 GSSTPSGATGSPGASP 455GGTAGCTCTACCCCGTCTGGTGCTACCG 456 GTSSTGSPGSSTPSGAGTTCCCCAGGTGCTTCTCCTGGTACTAG TGSP CTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_GFP-N_H10.ab1 GSSTPSGATGSPGTPG 457GGTAGCTCTACCCCGTCTGGTGCTACTG 458 SGTASSSPGSSTPSGAGTTCTCCAGGTACTCCGGGCAGCGGTAC TGSP TGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_GFP-N_A11.ab1 GASPGTSSTGSPGSSP 459GGTGCATCTCCTGGTACCAGCTCTACTG 460 SASTGTGPGSSTPSGAGTTCTCCAGGTTCTAGCCCTTCTGCTTCT TGSP ACCGGTACCGGTCCAGGTAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_GFP-N_B11.ab1 GSSTPSGATGSPGSST 461GGTAGCTCTACCCCGTCTGGTGCTACTG 462 PSGATGSPGSSTPSGAGCTCTCCAGGTAGCTCTACTCCTTCTGG TGSP TGCTACTGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCA LCW0404_052_GFP-N_C11.ab1 GASPGTSSTGSPGTPG 463GGTGCATCCCCGGGTACCAGCTCTACCG 464 SGTASSSPGASPGTSSGTTCTCCAGGTACTCCTGGCAGCGGTAC TGSP TGCATCTTCCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_GFP-N_D11.ab1 GSSTPSGATGSPGSSP 465GGTAGCTCTACTCCTTCTGGTGCAACTG 466 SASTGTGPGASPGTSSGTTCTCCAGGTTCTAGCCCGTCTGCATC TGSP CACTGGTACCGGTCCAGGTGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_057_GFP-N_E11.ab1 GASPGTSSTGSPGSST 467GGTGCATCTCCTGGTACTAGCTCTACTG 468 PSGATGSPGSSPSASTGTTCTCCAGGTAGCTCTACTCCGTCTGG GTGP TGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_GFP-N_F11.ab1 GTPGSGTASSSPGSST 469GGTACTCCTGGCAGCGGTACCGCATCTT 470 PSGATGSPGASPGTSSCCTCTCCAGGTAGCTCTACTCCGTCTGG TGSP TGCAACTGGTTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_GFP-N_G11.ab1 GSSTPSGATGSPGTPG 471GGTAGCTCTACCCCGTCTGGTGCAACCG 472 SGTASSSPGSSTPSGAGCTCCCCAGGTACTCCTGGTAGCGGTAC TGSP CGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_GFP-N_H11.ab1 GSSPSASTGTGPGSSP 473GGTTCTAGCCCTTCTGCATCCACCGGTA 474 SASTGTGPGASPGTSSCCGGCCCAGGTTCTAGCCCGTCTGCTTC TGSP TACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_GFP-N_A12.ab1 GTPGSGTASSSPGSST 475GGTACCCCGGGTAGCGGTACCGCTTCTT 476 PSGATGSPGSNPSASTCTTCTCCAGGTAGCTCTACTCCGTCTGG GTGP TGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCA LCW0404_068_GFP-N_B12.ab1 GSSPSASTGTGPGSST 477GGTTCTAGCCCTTCTGCATCTACTGGTA 478 PSGATGSPGASPGTSSCTGGCCCAGGTAGCTCTACTCCTTCTGG TGSP TGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_069_GFP-N_C12.ab1 GSSTPSGATGSPGASP 479GGTAGCTCTACCCCTTCTGGTGCAACCG 480 GTSSTGSPGTPGSGTAGCTCTCCAGGTGCATCCCCGGGTACCAG SSSP CTCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCA LCW0404_070_GFP-N_D12.ab1 GSSTPSGATGSPGSST 481GGTAGCTCTACTCCGTCTGGTGCAACCG 482 PSGATGSPGSSTPSGAGTTCCCCAGGTAGCTCTACCCCTTCTGG TGSP TGCAACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCA LCW0404_073_GFP-N_E12.ab1 GASPGTSSTGSPGTPG 483GGTGCTTCTCCTGGCACTAGCTCTACCG 484 SGTASSSPGSSTPSGAGTTCTCCAGGTACCCCTGGTAGCGGTAC TGSP CGCATCTTCCTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCA LCW0404_075_GFP-N_F12.ab1 GSSTPSGATGSPGSSP 485GGTAGCTCTACCCCGTCTGGTGCTACTG 486 SASTGTGPGSSPSASTGCTCCCCAGGTTCTAGCCCTTCTGCATC GTGP CACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCA LCW0404_080_GFP-N_G12.ab1 GASPGTSSTGSPGSSP 487GGTGCTTCCCCGGGCACCAGCTCTACTG 488 SASTGTGPGSSPSASTGTTCTCCAGGTTCTAGCCCGTCTGCTTCT GTGP ACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA LCW0404_081_GFP-N_H12.ab1 GASPGTSSTGSPGSSP 489GGTGCTTCCCCGGGTACCAGCTCTACCG 490 SASTGTGPGTPGSGTGTTCTCCAGGTTCTAGCCCTTCTGCTTCT ASSSP ACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA

Example 5: Construction of XTEN_AE864

XTEN_AE864 was constructed from serial dimerization of XTEN_AE36 toAE72, 144, 288, 576 and 864. A collection of XTEN_AE72 segments wasconstructed from 37 different segments of XTEN_AE36. Cultures of E. coliharboring all 37 different 36-amino acid segments were mixed and plasmidwas isolated. This plasmid pool was digested with BsaI/NcoI to generatethe small fragment as the insert. The same plasmid pool was digestedwith BbsI/NcoI to generate the large fragment as the vector. The insertand vector fragments were ligated resulting in a doubling of the lengthand the ligation mixture was transformed into BL21Gold(DE3) cells toobtain colonies of XTEN_AE72.

This library of XTEN_AE72 segments was designated LCW0406. All clonesfrom LCW0406 were combined and dimerized again using the same process asdescribed above yielding library LCW0410 of XTEN_AE144. All clones fromLCW0410 were combined and dimerized again using the same process asdescribed above yielding library LCW0414 of XTEN_AE288. Two isolatesLCW0414.001 and LCW0414.002 were randomly picked from the library andsequenced to verify the identities. All clones from LCW0414 werecombined and dimerized again using the same process as described aboveyielding library LCW0418 of XTEN_AE576. We screened 96 isolates fromlibrary LCW0418 for high level of GFP fluorescence. 8 isolates withright sizes of inserts by PCR and strong fluorescence were sequenced and2 isolates (LCW0418.018 and LCW0418.052) were chosen for future usebased on sequencing and expression data.

The specific clone pCW0432 of XTEN_AE864 was constructed by combiningLCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288 using the samedimerization process as described above.

Example 6: Construction of XTEN_AM144

A collection of XTEN_AM144 segments was constructed starting from 37different segments of XTEN_AE36, 44 segments of XTEN_AF36, and 44segments of XTEN_AG36.

Cultures of E. coli harboring all 125 different 36-amino acid segmentswere mixed and plasmid was isolated. This plasmid pool was digested withBsaI/NcoI to generate the small fragment as the insert. The same plasmidpool was digested with BbsI/NcoI to generate the large fragment as thevector. The insert and vector fragments were ligated resulting in adoubling of the length and the ligation mixture was transformed intoBL21Gold(DE3) cells to obtain colonies of XTEN_AM72.

This library of XTEN_AM72 segments was designated LCW0461. All clonesfrom LCW0461 were combined and dimerized again using the same process asdescribed above yielding library LCW0462. 1512 Isolates from libraryLCW0462 were screened for protein expression. Individual colonies weretransferred into 96 well plates and cultured overnight as startercultures. These starter cultures were diluted into fresh autoinductionmedium and cultured for 20-30 h. Expression was measured using afluorescence plate reader with excitation at 395 nm and emission at 510nm. 192 isolates showed high level expression and were submitted to DNAsequencing. Most clones in library LCW0462 showed good expression andsimilar physicochemical properties suggesting that most combinations ofXTEN_AM36 segments yield useful XTEN sequences. 30 isolates from LCW0462were chosen as a preferred collection of XTEN_AM144 segments for theconstruction of multifunctional proteins that contain multiple XTENsegments. The file names of the nucleotide and amino acid constructs forthese segments are listed in Table 13.

TABLE 13 DNA and amino acid sequences for AM144 se2ments SEQ SEQ ID IDClone Sequence Trimmed NO: Protein Sequence NO: LCW462_r1GGTACCCCGGGCAGCGGTACCGCATCTT 491 GTPGSGTASSSPGSSTPS 492CCTCTCCAGGTAGCTCTACCCCGTCTGGT GATGSPGSSTPSGATGSPGCTACCGGTTCCCCAGGTAGCTCTACCCC GSPAGSPTSTEEGTSESAGTCTGGTGCAACCGGCTCCCCAGGTAGC TPESGPGTSTEPSEGSAPCCGGCTGGCTCTCCTACCTCTACTGAGGA GSSPSASTGTGPGSSPSAAGGTACTTCTGAAAGCGCTACTCCTGAG STGTGPGASPGTSSTGSPTCTGGTCCAGGTACCTCTACTGAACCGTC GTSTEPSEGSAPGTSTEPCGAAGGTAGCGCTCCAGGTTCTAGCCCT SEGSAPGSEPATSGSETPTCTGCATCCACCGGTACCGGCCCAGGTTC TAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACT GGTTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACT GAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAAC TCCA LCW462_r5GGTTCTACCAGCGAATCCCCTTCTGGCAC 493 GSTSESPSGTAPGSTSESP 494TGCACCAGGTTCTACTAGCGAATCCCCTT SGTAPGTSPSGESSTAPGCTGGTACCGCACCAGGTACTTCTCCGAG TSTEPSEGSAPGTSTEPSECGGCGAATCTTCTACTGCTCCAGGTACCT GSAPGTSESATPESGPGACTACTGAACCTTCCGAAGGCAGCGCTCC SPGTSSTGSPGSSTPSGAAGGTACCTCTACCGAACCGTCCGAGGGC TGSPGASPGTSSTGSPGSAGCGCACCAGGTACTTCTGAAAGCGCAA TSESPSGTAPGSTSESPSGCCCCTGAATCCGGTCCAGGTGCATCTCCT TAPGTSTPESGSASPGGTACCAGCTCTACCGGTTCTCCAGGTA GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTAC CGGTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC GAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCT CCA LCW462_r9 GGTACTTCTACCGAACCTTCCGAGGGCA495 GTSTEPSEGSAPGTSESA 496 GCGCACCAGGTACTTCTGAAAGCGCTACTPESGPGTSESATPESGP CCCTGAGTCCGGCCCAGGTACTTCTGAA GTSTEPSEGSAPGTSESAAGCGCTACTCCTGAATCCGGTCCAGGTA TPESGPGTSTEPSEGSAPCCTCTACTGAACCTTCTGAGGGCAGCGCT GTSTEPSEGSAPGSEPATCCAGGTACTTCTGAAAGCGCTACCCCGG SGSETPGSPAGSPTSTEEAGTCCGGTCCAGGTACTTCTACTGAACC GASPGTSSTGSPGSSPSAGTCCGAAGGTAGCGCACCAGGTACTTCT STGTGPGSSPSASTGTGPACTGAACCTTCCGAAGGTAGCGCTCCAG GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGA CCTCCACCGAGGAAGGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAGGTTCTA GCCCTTCTGCTTCTACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGTAC TGGTCCA LCW462_r10GGTAGCGAACCGGCAACCTCTGGCTCTG 497 GSEPATSGSETPGTSESA 498AAACCCCAGGTACCTCTGAAAGCGCTAC TPESGPGTSESATPESGPTCCGGAATCTGGTCCAGGTACTTCTGAA GSTSESPSGTAPGSTSESPAGCGCTACTCCGGAATCCGGTCCAGGTT SGTAPGTSPSGESSTAPGCTACCAGCGAATCTCCTTCTGGCACCGCT ASPGTSSTGSPGSSPSASCCAGGTTCTACTAGCGAATCCCCGTCTGG TGTGPGSSTPSGATGSPGTACCGCACCAGGTACTTCTCCTAGCGGC SSTPSGATGSPGSSTPSGGAATCTTCTACCGCACCAGGTGCATCTCC ATGSPGASPGTSSTGSPGGGTACTAGCTCTACCGGTTCTCCAGGTT CTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTACCCCGTCTGGTGCTAC TGGTTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACT CCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTC CA LCW462_r15GGTGCTTCTCCGGGCACCAGCTCTACTGG 499 GASPGTSSTGSPGSSPSA 500TTCTCCAGGTTCTAGCCCTTCTGCATCCA STGTGPGSSTPSGATGSPCCGGTACCGGTCCAGGTAGCTCTACCCCT GTSESATPESGPGSEPATTCTGGTGCAACCGGCTCTCCAGGTACTTC SGSETPGSEPATSGSETPTGAAAGCGCTACCCCGGAATCTGGCCCA GTSESATPESGPGTSTEPGGTAGCGAACCGGCTACTTCTGGTTCTG SEGSAPGTSTEPSEGSAPAAACCCCAGGTAGCGAACCGGCTACCTC GTSTEPSEGSAPGTSTEPCGGTTCTGAAACTCCAGGTACTTCTGAA SEGSAPGSEPATSGSETPAGCGCTACTCCGGAGTCCGGTCCAGGTA CCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG GTAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCT ACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGA AACTCCA LCW462_r16GGTACCTCTACCGAACCTTCCGAAGGTA 501 GTSTEPSEGSAPGSPAGS 502GCGCTCCAGGTAGCCCGGCAGGTTCTCC PTSTEEGTSTEPSEGSAPTACTTCCACTGAGGAAGGTACTTCTACCG GTSESATPESGPGSEPATAACCTTCTGAGGGTAGCGCACCAGGTAC SGSETPGTSESATPESGPCTCTGAAAGCGCAACTCCTGAGTCTGGC GSPAGSPTSTEEGTSESACCAGGTAGCGAACCTGCTACCTCCGGCT TPESGPGTSTEPSEGSAPCTGAGACTCCAGGTACCTCTGAAAGCGC GSEPATSGSETPGTSTEPAACCCCGGAATCTGGTCCAGGTAGCCCG SEGSAPGSEPATSGSETPGCTGGCTCTCCTACCTCTACTGAGGAAG GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCG AAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCAGGTACTT CTACCGAACCGTCCGAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTG AAACTCCA LCW462_r20GGTACTTCTACCGAACCGTCCGAAGGCA 503 GTSTEPSEGSAPGTSTEP 504GCGCTCCAGGTACCTCTACTGAACCTTCC SEGSAPGTSTEPSEGSAPGAGGGCAGCGCTCCAGGTACCTCTACCG GTSTEPSEGSAPGTSTEPAACCTTCTGAAGGTAGCGCACCAGGTAC SEGSAPGTSTEPSEGSAPTTCTACCGAACCGTCCGAAGGCAGCGCT GTSTEPSEGSAPGTSESACCAGGTACCTCTACTGAACCTTCCGAGG TPESGPGTSESATPESGPGCAGCGCTCCAGGTACCTCTACCGAACC GTSTEPSEGSAPGSEPATTTCTGAAGGTAGCGCACCAGGTACTTCT SGSETPGSPAGSPTSTEEACCGAACCTTCCGAGGGCAGCGCACCAG GTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTC CTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCG AACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCA CCGAGGAA LCW462_r22GGTACTTCTACCGAACCGTCCGAGGGCA 505 GTSTEPSEGSAPGTSTEP 506GCGCTCCAGGTACTTCTACTGAACCTTCT SEGSAPGTSTEPSEGSAPGAAGGCAGCGCTCCAGGTACTTCTACTG GSTSESPSGTAPGSTSESPAACCTTCCGAAGGTAGCGCACCAGGTTC SGTAPGTSTPESGSASPGTACCAGCGAATCCCCTTCTGGTACTGCTC SEPATSGSETPGTSESATCAGGTTCTACCAGCGAATCCCCTTCTGGC PESGPGTSTEPSEGSAPGACCGCACCAGGTACTTCTACCCCTGAAA TSTEPSEGSAPGTSESATGCGGCTCCGCTTCTCCAGGTAGCGAACC PESGPGTSESATPESGPTGCAACCTCTGGCTCTGAAACCCCAGGT ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA GGGCAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTT CTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG TCCGGCCCA LCW462_r24GGTAGCTCTACCCCTTCTGGTGCTACCGG 507 GSSTPSGATGSPGSSPSA 508CTCTCCAGGTTCTAGCCCGTCTGCTTCTA STGTGPGSSTPSGATGSPCCGGTACCGGTCCAGGTAGCTCTACCCCT GSPAGSPTSTEEGSPAGSTCTGGTGCTACTGGTTCTCCAGGTAGCCC PTSTEEGTSTEPSEGSAPTGCTGGCTCTCCGACTTCTACTGAGGAAG GASPGTSSTGSPGSSPSAGTAGCCCGGCTGGTTCTCCGACTTCTACT STGTGPGTPGSGTASSSPGAGGAAGGTACTTCTACCGAACCTTCCG GSTSSTAESPGPGTSPSGAAGGTAGCGCTCCAGGTGCTTCCCCGGG ESSTAPGTSTPESGSASPCACTAGCTCTACCGGTTCTCCAGGTTCTA GCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTC CTCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTCCTAGC GGTGAATCTTCTACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA LCW462_r27 GGTACCTCTACTGAACCTTCTGAGGGCA 509GTSTEPSEGSAPGTSESA 510 GCGCTCCAGGTACTTCTGAAAGCGCTAC TPESGPGTSTEPSEGSAPCCCGGAGTCCGGTCCAGGTACTTCTACTG GTSTEPSEGSAPGTSESAAACCGTCCGAAGGTAGCGCACCAGGTAC TPESGPGTSESATPESGPTTCTACTGAACCGTCTGAAGGTAGCGCA GTPGSGTASSSPGASPGTCCAGGTACTTCTGAAAGCGCAACCCCGG SSTGSPGASPGTSSTGSPAATCCGGCCCAGGTACCTCTGAAAGCGC GSPAGSPTSTEEGSPAGSAACCCCGGAGTCCGGCCCAGGTACTCCT PTSTEEGTSTEPSEGSAPGGCAGCGGTACCGCTTCTTCTTCTCCAGG TGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCT ACTGGTTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCG GCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAG CGCTCCA LCW462_r28GGTAGCCCAGCAGGCTCTCCGACTTCCA 511 GSPAGSPTSTEEGTSTEP 512CTGAGGAAGGTACTTCTACTGAACCTTCC SEGSAPGTSTEPSEGSAPGAAGGCAGCGCACCAGGTACCTCTACTG GTSTEPSEGSAPGTSESAAACCTTCTGAGGGCAGCGCTCCAGGTAC TPESGPGTSESATPESGPCTCTACCGAACCGTCTGAAGGTAGCGCA GTPGSGTASSSPGSSTPSCCAGGTACCTCTGAAAGCGCAACTCCTG GATGSPGASPGTSSTGSPAGTCCGGTCCAGGTACTTCTGAAAGCGC GTSTEPSEGSAPGTSESAAACCCCGGAGTCTGGCCCAGGTACCCCG TPESGPGTSTEPSEGSAPGGTAGCGGTACTGCTTCTTCCTCTCCAGG TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCT ACCGGTTCTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTG AAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGC GCACCA LCW462_r38GGTAGCGAACCGGCAACCTCCGGCTCTG 513 GSEPATSGSETPGTSESA 514AAACTCCAGGTACTTCTGAAAGCGCTAC TPESGPGSEPATSGSETPTCCGGAATCCGGCCCAGGTAGCGAACCG GSSTPSGATGSPGTPGSGGCTACTTCCGGCTCTGAAACCCCAGGTA TASSSPGSSTPSGATGSPGCTCTACCCCGTCTGGTGCAACCGGCTCC GASPGTSSTGSPGSSTPSCCAGGTACTCCTGGTAGCGGTACCGCTTC GATGSPGASPGTSSTGSPTTCTTCTCCAGGTAGCTCTACTCCGTCTG GSEPATSGSETPGTSTEPGTGCTACCGGCTCCCCAGGTGCATCTCCT SEGSAPGSEPATSGSETPGGTACCAGCTCTACCGGTTCTCCAGGTA GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCTAC CGGTTCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACT CCA LCW462_r39GGTACCTCTACTGAACCTTCCGAAGGCA 515 GTSTEPSEGSAPGTSTEP 516GCGCTCCAGGTACCTCTACCGAACCGTC SEGSAPGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAA GSPAGSPTSTEEGSPAGSAGCGCAACCCCTGAATCCGGTCCAGGTA PTSTEEGTSTEPSEGSAPGCCCTGCTGGCTCTCCGACTTCTACTGAG GSPAGSPTSTEEGTSTEPGAAGGTAGCCCGGCTGGTTCTCCGACTT SEGSAPGTSTEPSEGSAPCTACTGAGGAAGGTACTTCTACCGAACC GASPGTSSTGSPGSSPSATTCCGAAGGTAGCGCTCCAGGTAGCCCG STGTGPGSSPSASTGTGPGCTGGTTCTCCGACTTCCACCGAGGAAG GTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACTGAACCTTCCGA AGGCAGCGCTCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAG CCCGTCTGCTTCTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACT GGTCCA LCW462_r41GGTAGCTCTACCCCGTCTGGTGCTACCGG 517 GSSTPSGATGSPGASPGT 518TTCCCCAGGTGCTTCTCCTGGTACTAGCT SSTGSPGSSTPSGATGSPCTACCGGTTCTCCAGGTAGCTCTACCCCG GSPAGSPTSTEEGTSESATCTGGTGCTACTGGCTCTCCAGGTAGCCC TPESGPGSEPATSGSETPTGCTGGCTCTCCAACCTCCACCGAAGAA GASPGTSSTGSPGSSTPSGGTACCTCTGAAAGCGCAACCCCTGAAT GATGSPGSSPSASTGTGPCCGGCCCAGGTAGCGAACCGGCAACCTC GSTSESPSGTAPGSTSESPCGGTTCTGAAACCCCAGGTGCATCTCCTG SGTAPGTSTPESGSASPGTACTAGCTCTACTGGTTCTCCAGGTAGC TCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTA CTGGTCCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGA ATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW462_r42 GGTTCTACCAGCGAATCTCCTTCTGGCAC519 GSTSESPSGTAPGSTSESP 520 CGCTCCAGGTTCTACTAGCGAATCCCCGTSGTAPGTSPSGESSTAPG CTGGTACCGCACCAGGTACTTCTCCTAGC TSESATPESGPGTSTEPSEGGCGAATCTTCTACCGCACCAGGTACCT GSAPGTSTEPSEGSAPGTCTGAAAGCGCTACTCCGGAGTCTGGCCC STEPSEGSAPGTSESATPAGGTACCTCTACTGAACCGTCTGAGGGT ESGPGTSTEPSEGSAPGSAGCGCTCCAGGTACTTCTACTGAACCGTC STPSGATGSPGASPGTSSCGAAGGTAGCGCACCAGGTACCTCTACT TGSPGSSTPSGATGSPGAACCTTCTGAGGGCAGCGCTCCAGGTA CTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAA GGTAGCGCACCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCT CCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCT CTCCA LCW462_r43GGTTCTACTAGCTCTACTGCAGAATCTCC 521 GSTSSTAESPGPGTSPSG 522GGGCCCAGGTACCTCTCCTAGCGGTGAA ESSTAPGTSPSGESSTAPTCTTCTACCGCTCCAGGTACTTCTCCGAG GSTSSTAESPGPGSTSSTCGGTGAATCTTCTACCGCTCCAGGTTCTA AESPGPGTSTPESGSASPCTAGCTCTACCGCTGAATCTCCGGGTCCA GTSPSGESSTAPGSTSSTGGTTCTACCAGCTCTACTGCAGAATCTCC AESPGPGTSTPESGSASPTGGCCCAGGTACTTCTACTCCGGAAAGC GSTSSTAESPGPGSTSESPGGTTCCGCTTCTCCAGGTACTTCTCCTAG SGTAPGTSPSGESSTAPCGGTGAATCTTCTACCGCTCCAGGTTCTA CCAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACCCCGGAAAGCGGCTCCG CTTCTCCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCG AATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCAC CA LCW462_r45 GGTACCTCTACTCCGGAAAGCGGTTCCG523 GTSTPESGSASPGSTSESP 524 CATCTCCAGGTTCTACCAGCGAATCCCCGSGTAPGSTSSTAESPGPG TCTGGCACCGCACCAGGTTCTACTAGCTC TSTEPSEGSAPGTSTEPSETACTGCTGAATCTCCGGGCCCAGGTACCT GSAPGTSESATPESGPGTCTACTGAACCTTCCGAAGGCAGCGCTCC SESATPESGPGTSTEPSEAGGTACCTCTACCGAACCGTCCGAGGGC GSAPGTSTEPSEGSAPGTAGCGCACCAGGTACTTCTGAAAGCGCAA SESATPESGPGTSTEPSECCCCTGAATCCGGTCCAGGTACCTCTGA GSAPGTSTEPSEGSAPAAGCGCTACTCCGGAGTCTGGCCCAGGT ACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAA GGTAGCGCACCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCT ACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGC GCTCCC LCW462_r47GGTACCTCTACCGAACCGTCCGAGGGTA 525 GTSTEPSEGSAPGTSTEP 526GCGCACCAGGTACCTCTACTGAACCGTC SEGSAPGSEPATSGSETPTGAGGGTAGCGCTCCAGGTAGCGAACCG GTSTEPSEGSAPGTSESAGCAACCTCCGGTTCTGAAACTCCAGGTA TPESGPGTSESATPESGPCTTCTACTGAACCGTCTGAAGGTAGCGC GASPGTSSTGSPGSSPSAACCAGGTACTTCTGAAAGCGCAACCCCG STGTGPGSSTPSGATGSPGAATCCGGCCCAGGTACCTCTGAAAGCG GSSTPSGATGSPGSSTPSCAACCCCGGAGTCCGGCCCAGGTGCATC GATGSPGASPGTSSTGSPTCCGGGTACTAGCTCTACCGGTTCTCCAG GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTACCCCGTCTGGTG CTACTGGTTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTC TACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGT TCTCCA LCW462_r54GGTAGCGAACCGGCAACCTCTGGCTCTG 527 GSEPATSGSETPGSEPAT 528AAACTCCAGGTAGCGAACCTGCAACCTC SGSETPGTSTEPSEGSAPCGGCTCTGAAACCCCAGGTACTTCTACTG GSEPATSGSETPGTSESAAACCTTCTGAGGGCAGCGCACCAGGTAG TPESGPGTSTEPSEGSAPCGAACCTGCAACCTCTGGCTCTGAAACC GSSTPSGATGSPGSSTPSCCAGGTACCTCTGAAAGCGCTACTCCTG GATGSPGASPGTSSTGSPAATCTGGCCCAGGTACTTCTACTGAACC GSSTPSGATGSPGASPGTGTCCGAGGGCAGCGCACCAGGTAGCTCT SSTGSPGSSTPSGATGSPACTCCGTCTGGTGCTACCGGCTCTCCAGG TAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTCTCCGGGTACCAGCTCT ACTGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTC CTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTC TCCA LCW462_r55GGTACTTCTACCGAACCGTCCGAGGGCA 529 GTSTEPSEGSAPGTSTEP 530GCGCTCCAGGTACTTCTACTGAACCTTCT SEGSAPGTSTEPSEGSAPGAAGGCAGCGCTCCAGGTACTTCTACTG GTSESATPESGPGTSTEPAACCTTCCGAAGGTAGCGCACCAGGTAC SEGSAPGTSTEPSEGSAPTTCTGAAAGCGCTACTCCGGAGTCCGGT GSTSESPSGTAPGTSPSGCCAGGTACCTCTACCGAACCGTCCGAAG ESSTAPGTSPSGESSTAPGCAGCGCTCCAGGTACTTCTACTGAACCT GSPAGSPTSTEEGTSESATCTGAGGGTAGCGCTCCAGGTTCTACTA TPESGPGTSTEPSEGSAPGCGAATCTCCGTCTGGCACTGCTCCAGGT ACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTT CTACCGCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG CTCCA LCW462_r57GGTACTTCTACTGAACCTTCCGAAGGTA 531 GTSTEPSEGSAPGSEPAT 532GCGCTCCAGGTAGCGAACCTGCTACTTCT SGSETPGSPAGSPTSTEEGGTTCTGAAACCCCAGGTAGCCCGGCTG GSPAGSPTSTEEGTSESAGCTCTCCGACCTCCACCGAGGAAGGTAG TPESGPGTSTEPSEGSAPCCCGGCAGGCTCTCCGACCTCTACTGAG GTSTEPSEGSAPGTSTEPGAAGGTACTTCTGAAAGCGCAACCCCGG SEGSAPGTSESATPESGPAGTCCGGCCCAGGTACCTCTACCGAACC GSSTPSGATGSPGSSPSAGTCTGAGGGCAGCGCACCAGGTACCTCT STGTGPGASPGTSSTGSPACTGAACCTTCCGAAGGCAGCGCTCCAG GTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACC CCTGAATCCGGTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTA GCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTG GTTCTCCA LCW462_r61GGTAGCGAACCGGCTACTTCCGGCTCTG 533 GSEPATSGSETPGSPAGS 534AGACTCCAGGTAGCCCTGCTGGCTCTCC PTSTEEGTSESATPESGPGACCTCTACCGAAGAAGGTACCTCTGAA GTSTEPSEGSAPGTSTEPAGCGCTACCCCTGAGTCTGGCCCAGGTA SEGSAPGTSESATPESGPCCTCTACTGAACCTTCCGAAGGCAGCGC GTSTPESGSASPGSTSESPTCCAGGTACCTCTACCGAACCGTCCGAG SGTAPGSTSSTAESPGPGGGCAGCGCACCAGGTACTTCTGAAAGCG TSESATPESGPGTSTEPSECAACCCCTGAATCCGGTCCAGGTACCTCT GSAPGTSTEPSEGSAPACTCCGGAAAGCGGTTCCGCATCTCCAG GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCTGA ATCTCCGGGCCCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCT CTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTA GCGCTCCA LCW462_r64GGTACTTCTACCGAACCGTCCGAGGGCA 535 GTSTEPSEGSAPGTSTEP 536GCGCTCCAGGTACTTCTACTGAACCTTCT SEGSAPGTSTEPSEGSAPGAAGGCAGCGCTCCAGGTACTTCTACTG GTSTEPSEGSAPGTSESAAACCTTCCGAAGGTAGCGCACCAGGTAC TPESGPGTSESATPESGPCTCTACCGAACCGTCTGAAGGTAGCGCA GTPGSGTASSSPGSSTPSCCAGGTACCTCTGAAAGCGCAACTCCTG GATGSPGASPGTSSTGSPAGTCCGGTCCAGGTACTTCTGAAAGCGC GSTSSTAESPGPGTSPSGAACCCCGGAGTCTGGCCCAGGTACTCCT ESSTAPGTSTPESGSASPGGCAGCGGTACCGCATCTTCCTCTCCAG GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTCTCCGGGTACCAGCTC TACCGGTTCTCCAGGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCT CCTAGCGGTGAATCTTCTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGCTT CTCCA LCW462_r67GGTAGCCCGGCAGGCTCTCCGACCTCTA 537 GSPAGSPTSTEEGTSESA 538CTGAGGAAGGTACTTCTGAAAGCGCAAC TPESGPGTSTEPSEGSAPCCCGGAGTCCGGCCCAGGTACCTCTACC GTSESATPESGPGSEPATGAACCGTCTGAGGGCAGCGCACCAGGTA SGSETPGTSTEPSEGSAPCTTCTGAAAGCGCAACCCCTGAATCCGG GSPAGSPTSTEEGTSTEPTCCAGGTAGCGAACCGGCTACTTCTGGC SEGSAPGTSTEPSEGSAPTCTGAGACTCCAGGTACTTCTACCGAACC GTSTEPSEGSAPGTSTEPGTCCGAAGGTAGCGCACCAGGTAGCCCG SEGSAPGTSTEPSEGSAPGCTGGTTCTCCGACTTCCACCGAGGAAG GTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACTGAACCTTCCGA AGGCAGCGCTCCAGGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACTT CTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAAGGT AGCGCACCA LCW462_r69GGTACTTCTCCGAGCGGTGAATCTTCTAC 539 GTSPSGESSTAPGSTSST 540CGCACCAGGTTCTACTAGCTCTACCGCTG AESPGPGTSPSGESSTAPAATCTCCGGGCCCAGGTACTTCTCCGAG GTSESATPESGPGTSTEPCGGTGAATCTTCTACTGCTCCAGGTACCT SEGSAPGTSTEPSEGSAPCTGAAAGCGCTACTCCGGAGTCTGGCCC GSSPSASTGTGPGSSTPSAGGTACCTCTACTGAACCGTCTGAGGGT GATGSPGASPGTSSTGSPAGCGCTCCAGGTACTTCTACTGAACCGTC GTSTPESGSASPGTSPSGCGAAGGTAGCGCACCAGGTTCTAGCCCT ESSTAPGTSPSGESSTAPTCTGCATCTACTGGTACTGGCCCAGGTAG CTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACC GGTTCTCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTA GCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCC A LCW462_r70 GGTACCTCTGAAAGCGCTACTCCGGAGT541 GTSESATPESGPGTSTEP 542 CTGGCCCAGGTACCTCTACTGAACCGTCTSEGSAPGTSTEPSEGSAP GAGGGTAGCGCTCCAGGTACTTCTACTG GSPAGSPTSTEEGSPAGSAACCGTCCGAAGGTAGCGCACCAGGTAG PTSTEEGTSTEPSEGSAPCCCTGCTGGCTCTCCGACTTCTACTGAGG GSSPSASTGTGPGSSTPSAAGGTAGCCCGGCTGGTTCTCCGACTTCT GATGSPGSSTPSGATGSPACTGAGGAAGGTACTTCTACCGAACCTT GSEPATSGSETPGTSESACCGAAGGTAGCGCTCCAGGTTCTAGCCC TPESGPGSEPATSGSETPTTCTGCTTCCACCGGTACTGGCCCAGGTA GCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAAC TGGCTCTCCAGGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGA AAGCGCTACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAA CCCCA LCW462_r72GGTACTTCTACCGAACCGTCCGAAGGCA 543 GTSTEPSEGSAPGTSTEP 544GCGCTCCAGGTACCTCTACTGAACCTTCC SEGSAPGTSTEPSEGSAPGAGGGCAGCGCTCCAGGTACCTCTACCG GSSTPSGATGSPGASPGTAACCTTCTGAAGGTAGCGCACCAGGTAG SSTGSPGSSTPSGATGSPCTCTACCCCGTCTGGTGCTACCGGTTCCC GTSESATPESGPGSEPATCAGGTGCTTCTCCTGGTACTAGCTCTACC SGSETPGTSTEPSEGSAPGGTTCTCCAGGTAGCTCTACCCCGTCTGG GSTSESPSGTAPGSTSESPTGCTACTGGCTCTCCAGGTACTTCTGAAA SGTAPGTSTPESGSASPGCGCAACCCCTGAATCCGGTCCAGGTAG CGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGG TAGCGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAG CGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTC TCCA LCW462_r73GGTACCTCTACTCCTGAAAGCGGTTCTGC 545 GTSTPESGSASPGSTSST 546ATCTCCAGGTTCCACTAGCTCTACCGCAG AESPGPGSTSSTAESPGPAATCTCCGGGCCCAGGTTCTACTAGCTCT GSSPSASTGTGPGSSTPSACTGCTGAATCTCCTGGCCCAGGTTCTAG GATGSPGASPGTSSTGSPCCCTTCTGCATCTACTGGTACTGGCCCAG GSEPATSGSETPGTSESAGTAGCTCTACTCCTTCTGGTGCTACCGGC TPESGPGSPAGSPTSTEETCTCCAGGTGCTTCTCCGGGTACTAGCTC GSTSESPSGTAPGSTSESPTACCGGTTCTCCAGGTAGCGAACCGGCA SGTAPGTSTPESGSASPACCTCCGGCTCTGAAACCCCAGGTACCT CTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCC ACTGAGGAAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC GAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCT CCC LCW462_r78GGTAGCCCGGCTGGCTCTCCTACCTCTAC 547 GSPAGSPTSTEEGTSESA 548TGAGGAAGGTACTTCTGAAAGCGCTACT TPESGPGTSTEPSEGSAPCCTGAGTCTGGTCCAGGTACCTCTACTGA GSTSESPSGTAPGSTSESPACCGTCCGAAGGTAGCGCTCCAGGTTCT SGTAPGTSPSGESSTAPGACCAGCGAATCTCCTTCTGGCACCGCTCC TSTEPSEGSAPGSPAGSPAGGTTCTACTAGCGAATCCCCGTCTGGTA TSTEEGTSTEPSEGSAPGCCGCACCAGGTACTTCTCCTAGCGGCGA SEPATSGSETPGTSESATATCTTCTACCGCACCAGGTACCTCTACCG PESGPGTSTEPSEGSAPAACCTTCCGAAGGTAGCGCTCCAGGTAG CCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCGAACCTTCTGAGGG TAGCGCACCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGA AAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA LCW462_r79GGTACCTCTACCGAACCTTCCGAAGGTA 549 GTSTEPSEGSAPGSPAGS 550GCGCTCCAGGTAGCCCGGCAGGTTCTCC PTSTEEGTSTEPSEGSAPTACTTCCACTGAGGAAGGTACTTCTACCG GTSPSGESSTAPGTSPSGAACCTTCTGAGGGTAGCGCACCAGGTAC ESSTAPGTSPSGESSTAPCTCCCCTAGCGGCGAATCTTCTACTGCTC GSTSESPSGTAPGSTSESPCAGGTACCTCTCCTAGCGGCGAATCTTCT SGTAPGTSTPESGSASPGACCGCTCCAGGTACCTCCCCTAGCGGTG SEPATSGSETPGTSESATAATCTTCTACCGCACCAGGTTCTACCAGC PESGPGTSTEPSEGSAPGAATCCCCTTCTGGTACTGCTCCAGGTTC TACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCT CCGCTTCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG AAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA LCW462_r87GGTAGCGAACCGGCAACCTCTGGCTCTG 551 GSEPATSGSETPGTSESA 552AAACCCCAGGTACCTCTGAAAGCGCTAC TPESGPGTSESATPESGPTCCGGAATCTGGTCCAGGTACTTCTGAA GTSPSGESSTAPGSTSSTAGCGCTACTCCGGAATCCGGTCCAGGTA AESPGPGTSPSGESSTAPCTTCTCCGAGCGGTGAATCTTCTACCGCA GSTSESPSGTAPGTSPSGCCAGGTTCTACTAGCTCTACCGCTGAATC ESSTAPGSTSSTAESPGPTCCGGGCCCAGGTACTTCTCCGAGCGGT GSSTPSGATGSPGSSTPSGAATCTTCTACTGCTCCAGGTTCTACTAG GATGSPGSSTPSGANWLCGAATCCCCGTCTGGTACTGCTCCAGGTA S CTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCAGAAT CTCCGGGTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTA CCCCTTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACTGGCT CTCC LCW462_r88GGTAGCCCTGCTGGCTCTCCGACTTCTAC 553 GSPAGSPTSTEEGSPAGS 554TGAGGAAGGTAGCCCGGCTGGTTCTCCG PTSTEEGTSTEPSEGSAPACTTCTACTGAGGAAGGTACTTCTACCG GTSTEPSEGSAPGTSTEPAACCTTCCGAAGGTAGCGCTCCAGGTAC SEGSAPGTSESATPESGPCTCTACTGAACCTTCCGAAGGCAGCGCT GASPGTSSTGSPGSSTPSCCAGGTACCTCTACCGAACCGTCCGAGG GATGSPGASPGTSSTGSPGCAGCGCACCAGGTACTTCTGAAAGCGC GSSTPSGATGSPGTPGSGAACCCCTGAATCCGGTCCAGGTGCATCT TASSSPGSSTPSGATGSPCCTGGTACCAGCTCTACCGGTTCTCCAGG TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCCGGGTACCAGCTCT ACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGG GCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTC TCCA LCW462_r89GGTAGCTCTACCCCGTCTGGTGCTACTGG 555 GSSTPSGATGSPGTPGSG 556TTCTCCAGGTACTCCGGGCAGCGGTACT TASSSPGSSTPSGATGSPGCTTCTTCCTCTCCAGGTAGCTCTACCCC GSPAGSPTSTEEGTSESATTCTGGTGCTACTGGCTCTCCAGGTAGCC TPESGPGTSTEPSEGSAPCGGCTGGCTCTCCTACCTCTACTGAGGAA GTSESATPESGPGSEPATGGTACTTCTGAAAGCGCTACTCCTGAGTC SGSETPGTSESATPESGPTGGTCCAGGTACCTCTACTGAACCGTCCG GTSTEPSEGSAPGTSESAAAGGTAGCGCTCCAGGTACCTCTGAAAG TPESGPGTSESATPESGPCGCAACTCCTGAGTCTGGCCCAGGTAGC GAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAA TCTGGTCCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAA AGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGG CCCA

Example 7: Construction of XTEN_AM288

The entire library LCW0462 was dimerized as described in Example 6resulting in a library of XTEN_AM288 clones designated LCW0463. 1512isolates from library LCW0463 were screened using the protocol describedin Example 6. 176 highly expressing clones were sequenced and 40preferred XTEN_AM288 segments were chosen for the construction ofmultifunctional proteins that contain multiple XTEN segments with 288amino acid residues.

Example 8: Construction of XTEN_AM432

We generated a library of XTEN_AM432 segments by recombining segmentsfrom library LCW0462 of XTEN_AM144 segments and segments from libraryLCW0463 of XTEN_AM288 segments. This new library of XTEN_AM432 segmentwas designated LCW0464. Plasmid was isolated from cultures of E. coliharboring LCW0462 and LCW0463, respectively. 1512 isolates from libraryLCW0464 were screened using the protocol described in Example 6. 176highly expressing clones were sequenced and 39 preferred XTEN_AM432segment were chosen for the construction of longer XTENs and for theconstruction of multifunctional proteins that contain multiple XTENsegments with 432 amino acid residues.

In parallel we constructed library LMS0100 of XTEN_AM432 segments usingpreferred segments of XTEN_AM144 and XTEN_AM288. Screening of thislibrary yielded 4 isolates that were selected for further construction.

Example 9: Construction of XTEN_AM875

The stuffer vector pCW0359 was digested with BsaI and KpnI to remove thestuffer segment and the resulting vector fragment was isolated byagarose gel purification.

We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnIforP:AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO:557) and the non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev:CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 558) forintroducing the sequencing island A (SI-A) which encodes amino acidsGASASGAPSTG (SEQ ID NO: 559) and has the restriction enzyme AscIrecognition nucleotide sequence GGCGCGCC inside. The annealedoligonucleotide pairs were ligated with BsaI and KpnI digested stuffervector pCW0359 prepared above to yield pCW0466 containing SI-A. We thengenerated a library of XTEN_AM443 segments by recombining 43 preferredXTEN_AM432 segments from Example 8 and SI-A segments from pCW0466 atC-terminus using the same dimerization process described in Example 5.This new library of XTEN_AM443 segments was designated LCW0479.

We generated a library of XTEN_AM875 segments by recombining segmentsfrom library LCW0479 of XTEN_AM443 segments and 43 preferred XTEN_AM432segments from Example 8 using the same dimerization process described inExample 5. This new library of XTEN_AM875 segment was designatedLCW0481.

Example 10: Construction of XTEN_AM1318

We annealed the phosphorylated oligonucleotide BsaI-FseI-KpnIforP:AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO:560) and the non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev:CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 561) forintroducing the sequencing island B (SI-B) which encodes amino acidsGPEPTGPAPSG (SEQ ID NO: 562) and has the restriction enzyme FseIrecognition nucleotide sequence GGCCGGCC inside. The annealedoligonucleotide pairs were ligated with BsaI and KpnI digested stuffervector pCW0359 as used in Example 9 to yield pCW0467 containing SI-B. Wethen generated a library of XTEN_AM443 segments by recombining 43preferred XTEN_AM432 segments from Example 8 and SI-B segments frompCW0467 at C-terminus using the same dimerization process described inExample 5. This new library of XTEN_AM443 segments was designatedLCW0480.

We generated a library of XTEN_AM1318 segments by recombining segmentsfrom library LCW0480 of XTEN_AM443 segments and segments from libraryLCW0481 of XTEN_AM875 segments using the same dimerization process as inExample 5. This new library of XTEN_AM1318 segment was designatedLCW0487.

Example 11: Construction of XTEN_AD864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AD864 sequences starting from segments of XTEN_AD36listed in Example 1. These sequences were assembled as described inExample 5. Several isolates from XTEN_AD864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. One intermediate construct of XTEN_AD576 was sequenced. Thisclone was evaluated in a PK experiment in cynomolgus monkeys and ahalf-life of about 20 h was measured.

Example 12: Construction of XTEN_AF864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AF864 sequences starting from segments of XTEN_AF36listed in Example 3. These sequences were assembled as described inExample 5. Several isolates from XTEN_AF864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. One intermediate construct of XTEN_AF540 was sequenced. Thisclone was evaluated in a PK experiment in cynomolgus monkeys and ahalf-life of about 20 h was measured. A full length clone of XTEN_AF864had excellent solubility and showed half-life exceeding 60 h incynomolgus monkeys. A second set of XTEN_AF sequences was assembledincluding a sequencing island as described in Example 9.

Example 13: Construction of XTEN_AG864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AG864 sequences starting from segments of XTEN_AD36listed in Example 1. These sequences were assembled as described inExample 5. Several isolates from XTEN_AG864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. A full-length clone of XTEN_AG864 had excellent solubilityand showed half-life exceeding 60 h in cynomolgus monkeys.

Example 14: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of 12mer Addition Libraries

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of fusion proteins withoutthe presence of a helper domain. Historically expression of proteinswith XTEN at the N-terminus was poor, yielding values that wouldessentially undetectable in the GFP fluorescence assay (<25% of theexpression with the N-terminal CBD helper domain). To create diversityat the codon level, seven amino acid sequences were selected andprepared with a diversity of codons. Seven pairs of oligonucleotidesencoding 12 amino acids with codon diversities were designed, annealedand ligated into the NdeI/BsaI restriction enzyme digested stuffervector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed into E. coliBL21Gold(DE3) competent cells to obtain colonies of seven libraries. Theresulting clones have N-terminal XTEN 12mers fused in-frame toXTEN_AM875-GFP to allow use of GFP fluorescence for screening theexpression. Individual colonies from the seven created libraries werepicked and grown overnight to saturation in 500 μl of super broth mediain a 96 deep well plate. The number of colonies picked ranged fromapproximately half to a third of the theoretical diversity of thelibrary (see Table 14).

TABLE 14Theoretical Diversity and Sampling Numbers for 12mer Addition Libraries.The amino acid residues with randomized codons are underlined.Amino Acid SEQ Theoretical Number Library Motif Family Sequence ID NO:Diversity screened LCW546 AE12 MASPAGSPTSTEE 563 572 2 plates (168)LCW547 AE12 MATSESATPESGP 564 1536 5 plates (420) LCW548 AF12MATSPSGESSTAP 565 192 2 plates (168) LCW549 AF12 MESTSSTAESPGP 566 3842 plates (168) LCW552 AG12 MASSTPSGATGSP 567 384 2 plates (168) LCW553AG12 MEASPGTSSTGSP 568 384 2 plates (168) LCW554 (CBD-like) MASTPESGSSG569 32 1 plate (84)

The saturated overnight cultures were used to inoculate fresh 500 μlcultures in auto-induction media in which they were grown overnight at26° C. These expression cultures were then assayed using a fluorescenceplate reader (excitation 395 nm, emission 510 nm) to determine theamount of GFP reporter present (see FIG. 28 for results of expressionassays). The results indicated that while median expression levels wereapproximately half of the expression levels compared to the “benchmark”CBD N-terminal helper domain, the best clones from the libraries weremuch closer to the benchmarks, indicating that further optimizationaround those sequences was warranted. This is in contrast to previousXTEN versions that were <25% of the expression levels of the CBDN-terminal benchmark. The results also show that the libraries startingwith amino acids MA had better expression levels than those beginningwith ME. This was most apparent when looking at the best clones, whichwere closer to the benchmarks as they mostly start with MA. Of the 176clones within 33% of the CBD-AM875 benchmark, 87% begin with MA, whereas only 75% of the sequences in the libraries beginning with MA, a clearover representation of the clones beginning with MA at the highest levelof expression. 96 of the best clones were sequenced to confirm identityand twelve sequences (see Table 15), 4 from LCW546, 4 from LCW547 and 4from LCW552 were selected for further optimization.

TABLE 15 Advanced 12mer DNA Nucleotide Sequences SEQ CloneDNA Nucleotide Sequence ID NO: LCW546_02ATGGCTAGTCCGGCTGGCTCTCCGACCTCCACTGAGGAAGGTACTTCTACT 570 LCW546_06ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACTTCTACT 571 LCW546_07ATGGCTAGTCCAGCAGGCTCTCCTACCTCCACCGAGGAAGGTACTTCTACT 572 LCW546_09ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTACT 573 LCW547_03ATGGCTACATCCGAAAGCGCAACCCCTGAGTCCGGTCCAGGTACTTCTACT 574 LCW547_06ATGGCTACATCCGAAAGCGCAACCCCTGAATCTGGTCCAGGTACTTCTACT 575 LCW547_10ATGGCTACGTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTACT 576 LCW547_17ATGGCTACGTCCGAAAGCGCTACCCCTGAATCCGGTCCAGGTACTTCTACT 577 LCW552_03ATGGCTAGTTCTACCCCGTCTGGTGCAACCGGTTCCCCAGGTACTTCTACT 578 LCW552_05ATGGCTAGCTCCACTCCGTCTGGTGCTACCGGTTCCCCAGGTACTTCTACT 579 LCW552_10ATGGCTAGCTCTACTCCGTCTGGTGCTACTGGTTCCCCAGGTACTTCTACT 580 LCW552_11ATGGCTAGTTCTACCCCTTCTGGTGCTACTGGTTCTCCAGGTACTTCTACT 581

Example 15: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Libraries Optimizing Codons 3 and 4

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first two codonsestablished (see Example supra), the third and fourth codons wererandomized to determine preferences. Three libraries, based upon bestclones from LCW546, LCW547 and LCW552, were designed with the third andfourth residues modified such that all combinations of allowable XTENcodons were present at these positions (see FIG. 29). In order toinclude all the allowable XTEN codons for each library, nine pairs ofoligonucleotides encoding 12 amino acids with codon diversities of thirdand fourth residues were designed, annealed and ligated into theNdeI/BsaI restriction enzyme digested stuffer vector pCW0551(Stuffer-XTEN_AM875-GFP), and transformed into E. coli BL21Gold(DE3)competent cells to obtain colonies of three libraries LCW0569-571. With24 XTEN codons the theoretical diversity of each library is 576 uniqueclones. A total of 504 individual colonies from the three createdlibraries were picked and grown overnight to saturation in 500 μl ofsuper broth media in a 96 deep well plate. This provided sufficientcoverage to understand relative library performance and sequencepreferences. The saturated overnight cultures were used to inoculate new500 μl cultures in auto-induction media in which were grown overnight at26° C. These expression cultures were then assayed using a fluorescenceplate reader (excitation 395 nm, emission 510 nm) to determine theamount of GFP reporter present. The top 75 clones from the screen weresequenced and retested for GFP reporter expression versus the benchmarksamples (see FIG. 28). 52 clones yielded usable sequencing data and wereused for subsequent analysis. The results were broken down by libraryand indicate that LCW546 was the superior library. The results arepresented in Table 16. Surprisingly, it was discovered that base-linedfluorescence readings for the best clones were ˜900 AU, whereas the CBDN-terminal benchmark was only ˜600 AU. This indicates that this libraryhad instituted an approximately 33% improvement over the best clonesfrom the previous library which were approximately equal in expressionto the CBD N-terminal benchmark (Example 14).

TABLE 16 Third and Fourth Codon Optimization Library Comparison LCW569LCW570 LCW571 N 21 15 16 Mean 628 491 537 Fluorescence (AU) SD 173 71232 CV 28% 15% 43%

Further trends were seen in the data showing preferences for particularcodons at the third and fourth position. Within the LCW569 library theglutamate codon GAA at the third position and the threonine codon ACTwere associated with higher expression as seen in Table 17.

TABLE 17 Preferred Third and Fourth Codons in LCW569 3 = GAA Rest 4 =ACT Rest N 8 13 4 17 Mean 749 554 744 601 Fluorescence (AU) SD 234 47197 162 CV 31% 9% 26% 27%

Additionally, the retest of the top 75 clones indicated that severalwere now superior to the benchmark clones.

Example 16: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Combinatorial 12mer and 36mer Libraries

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first two codonsestablished (see Example supra), the N-terminus was examined in abroader context by combining the 12 selected 12mer sequences (seeExample supra) at the very N-terminus followed by 125 previouslyconstructed 36mer segments (see example supra) in a combinatorialmanner. This created novel 48mers at the N-terminus of the XTEN proteinand enabled the assessment of the impact of longer-range interactions atthe N-terminus on expression of the longer sequences (FIG. 29). Similarto the dimerization procedures used to assemble 36mers (see Exampleinfra), the plasmids containing the 125 selected 36mer segments weredigested with restriction enzymes BbsI/NcoI and the appropriate fragmentwas gel-purified. The plasmid from clone AC94 (CBD-XTEN_AM875-GFP) wasalso digested with BsaI/NcoI and the appropriate fragments weregel-purified. These fragments were ligated together and transformed intoE. coli BL21Gold(DE3) competent cells to obtain colonies of the libraryLCW0579, which also served as the vector for further cloning 12 selected12mers at the very N-terminus. The plasmids of LCW0579 were digestedwith NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. 12pairs of oligonucleotides encoding 12 selected 12mer sequences weredesigned, annealed and ligated with the NdeI/EcoRI/BsaI digested LCW0579vector, and transformed into E. coli BL21Gold(DE3) competent cells toobtain colonies of the library LCW0580. With a theoretical diversity of1500 unique clones, a total of 1512 individual colonies from the createdlibrary were picked and grown overnight to saturation in 500 μl of superbroth media in a 96 deep well plate. This provided sufficient coverageto understand relative library performance and sequence preferences. Thesaturated overnight cultures were used to inoculate new 500 μl culturesin auto-induction media that were grown overnight at 26° C. Theseexpression cultures were then assayed using a fluorescence plate reader(excitation 395 nm, emission 510 nm) to determine the amount of GFPreporter present. The top 90 clones were sequenced and retested for GFPreporter expression. 83 clones yielded usable sequencing data and wereused for subsequent analysis. The sequencing data was used to determinethe lead 12mer that was present in each clone and the impact of each12mer on expression was assessed. Clones LCW546_06 and LCW546_09 stoodout as being the superior N-terminus (see Table 18).

TABLE 18 Relative Performance of Clones Starting with LCW546_06 andLCW459_09 All All LCW546_06 Others LCW546_09 Others N 11 72 9 74 Mean1100 752 988 775 Fluorescence (AU) SD 275 154 179 202 CV 25% 20% 18% 26%

The sequencing and retest also revealed several instances of independentreplicates of the same sequence in the data producing similar results,thus increasing confidence in the assay. Additionally, 10 clones with 6unique sequences were superior to the benchmark clone. They arepresented in Table 19. It was noted that these were the only occurrencesof these sequences and in no case did one of these sequences occur andfail to beat the bench-mark clone. These six sequences were advanced forfurther optimization.

TABLE 19Combinatorial 12mer and 36mer Clones Superior to Benchmark Clone CloneSEQ Name First 60 codons ID NO: 12mer Name 36mer Name LCW580_51ATGGCTAGTCCTGCTGGCTCTCCAACCTC 582 LCW546_06 LCW0404_040CACTGAGGAAGGTGCATCCCCGGGCACC AGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTA GCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCA LCW580_81ATGGCTAGTCCTGCTGGCTCTCCAACCTC 583 LCW546_06 LCW0404_040CACTGAGGAAGGTGCATCCCCGGGCACC AGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTA GCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCA LCW580_38ATGGCTAGTCCTGCTGGCTCTCCAACCTC 584 LCW546_06 LCW0402_041CACTGAGGAAGGTACTTCTACCGAACCGT CCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTA CTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGG CAGCGCA LCW580_63ATGGCTAGTCCTGCTGGCTCTCCGACCTC 585 LCW546_09 LCW0402_020TACTGAGGAAGGTACTTCTACTGAACCGT CTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTA GCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTACTTCTACTGAACCGTCTGAAGG CAGCGCA LCW580_06ATGGCTAGTCCTGCTGGCTCTCCAACCTC 586 LCW546_06 LCW0404_031CACTGAGGAAGGTACCCCGGGTAGCGGT ACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC TTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCA LCW580_35ATGGCTAGTCCTGCTGGCTCTCCGACCTC 587 LCW546_09 LCW0402_020TACTGAGGAAGGTACTTCTACTGAACCGT CTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTA GCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTACTTCTACTGAACCGTCTGAAGG CAGCGCA LCW580_67ATGGCTAGTCCTGCTGGCTCTCCGACCTC 588 LCW546_09 LCW0403_064TACTGAGGAAGGTACCTCCCCTAGCGGCG AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACC TCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACTGAACCGTCTGAAGGCA GCGCA LCW580_13ATGGCTAGTCCTGCTGGCTCTCCGACCTC 589 LCW546_09 LCW0403_060TACTGAGGAAGGTACCTCTACTCCGGAAA GCGGTTCCGCATCTCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTC TACTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCA LCW580_88ATGGCTAGTCCTGCTGGCTCTCCGACCTC 590 LCW546_09 LCW0403_064TACTGAGGAAGGTACCTCCCCTAGCGGCG AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACC TCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACTGAACCGTCTGAAGGCA GCGCA LCW580_11ATGGCTAGTCCTGCTGGCTCTCCGACCTC 591 LCW546_09 LCW0403_060TACTGAGGAAGGTACCTCTACTCCGGAAA GCGGTTCCGCATCTCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTC TACTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCA

Example 17: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Combinatorial 12mer and 36mer Libraries for XTEN-AM875and XTEN_AE864

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first four codons(see Examples supra, and for the best pairing of N-terminal 12mers and36mers (see Example supra) established, a combinatorial approach wasundertaken to examine the union of these preferences. This created novel48mers at the N-terminus of the XTEN protein and enabled the testing ofthe confluence of previous conclusions. Additionally, the ability ofthese leader sequences to be a universal solution for all XTEN proteinswas assessed by placing the new 48mers in front of both XTEN-AE864 andXTEN-AM875. Instead of using all 125 clones of 36mer segment, theplasmids from 6 selected clones of 36mer segment with best GFPexpression in the combinatorial library were digested withNdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. Theplasmids from clones AC94 (CBD-XTEN_AM875-GFP) and AC104(CBD-XTEN_AE864-GFP) were digested with digested with NdeI/EcoRI/BsaIand the appropriate fragments were gel-purified. These fragments wereligated together and transformed into E. coli BL21Gold(DE3) competentcells to obtain colonies of the libraries LCW0585 (-XTEN_AM875-GFP) andLCW0586 (-XTEN_AE864-GFP), which could also serve as the vectors forfurther cloning 8 selected 12mers at the very N-terminus. The plasmidsof LCW0585 and LCW0586 were digested with NdeI/EcoRI/BsaI and theappropriate fragments were gel-purified. 8 pairs of oligonucleotidesencoding 8 selected 12mer sequences with best GFP expression in theprevious (Generation 2) screening were designed, annealed and ligatedwith the NdeI/EcoRI/BsaI digested LCW0585 and LCW0586 vectors, andtransformed into E. coli BL21Gold(DE3) competent cells to obtaincolonies of the final libraries LCW0587 (XTEN_AM923-GFP) and LCW0588(XTEN_AE912-GFP). With a theoretical diversity of 48 unique clones, atotal of 252 individual colonies from the created libraries were pickedand grown overnight to saturation in 500 μl of super broth media in a 96deep well plate. This provided sufficient coverage to understandrelative library performance and sequence preferences. The saturatedovernight cultures were used to inoculate new 500 μl cultures inauto-induction media in which were grown overnight at 26° C. Theseexpression cultures were then assayed using a fluorescence plate reader(excitation 395 nm, emission 510 nm) to determine the amount of GFPreporter present. The top 36 clones were sequenced and retested for GFPreporter expression. 36 clones yielded usable sequencing data and these36 were used for the subsequent analysis. The sequencing data determinedthe 12mer, the third codon, the fourth codon and the 36mer present inthe clone and revealed that many of the clones were independentreplicates of the same sequence. Additionally, the retest results forthese clones are close in value, indicating the screening process wasrobust. Preferences for certain combinations at the N-terminus were seenand were consistently yielding higher fluorescence values approximately50% greater than the benchmark controls (see Tables 20 and 21). Thesedate support the conclusion that the inclusion of the sequences encodingthe optimized N-terminal XTEN into the fusion protein genes conferred amarked enhancement on the expression of the fusion proteins.

TABLE 20 Preferred N-terminal Combinations for XTEN-AM875 Number ofClone Name Replicates 12mer 36mer Mean SD CV CBD-AM875 NA NA NA 1715 41816% LCW587_08 7 LCW546_06_3 = GAA LCW404_40 2333 572 18% LCW587_17 5LCW546_09_3 = GAA LCW403_64 2172 293 10%

TABLE 21 Preferred N-terminal Combinations for XTEN-AE864 Number ofClone Name Replicates 12mer 36mer Mean SD CV AC82 NA NA NA 1979 679 24%LCW588_14 8 LCW546_06_opt3 LCW404_31 2801 240  6% LCW588_27 2LCW546_06_opt34 LCW404_40 2839 556 15%

Notably, the preferred combination of the N-terminal for the XTEN-AM875and the preferred combination for the XTEN-AE864 are not the same,indicating more complex interactions further than 150 bases from theinitiation site influence expression levels. The sequences for thepreferred nucleotide sequences are listed in Table 22 and the preferredclones were analyzed by SDS-PAGE to independently confirm expression(see FIG. 30). The complete sequences of XTEN_AM923 and XTEN_AE912 wereselected for further analysis.

TABLE 22 Preferred DNA Nucleotide Sequences for first 48 Amino AcidResidues of N-terminal XTEN-AM875 and XTEN-AE864  SEQ Clone XTEN ID NameModified DNA Nucleotide Sequence NO: LCW587_08 AM875ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCA 592TCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW587_17 AM875ATGGCTGAACCTGCTGGCTCTCCGACCTCTACTGAGGAAGGTACC 593TCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW588_14 AE864ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACC 594CCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG LCW588_27 AE864ATGGCTGAAACTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCA 595TCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG

Example 18: Methods of Producing and Evaluating CFXTEN; XTEN-CF asExample

A general schema for producing and evaluating CFXTEN compositions ispresented in FIG. 33, and forms the basis for the general description ofthis Example. Using the disclosed methods and those known to one ofordinary skill in the art, together with guidance provided in theillustrative examples, a skilled artesian can create and evaluate arange of CFXTEN fusion proteins comprising, XTENs, CF and variants of CFknown in the art. The Example is, therefore, to be construed as merelyillustrative, and not limitative of the methods in any way whatsoever;numerous variations will be apparent to the ordinarily skilled artisan.In this Example, a CFXTEN of coagulation factor linked to an XTEN of theAE family of motifs is created.

The general scheme for producing polynucleotides encoding XTEN ispresented in FIGS. 31 and 32. FIG. 32 is a schematic flowchart ofrepresentative steps in the assembly of a XTEN polynucleotide constructin one of the embodiments of the invention. Individual oligonucleotides501 are annealed into sequence motifs 502 such as a 12 amino acid motif(“12-mer”), which is subsequently ligated with an oligo containing BbsI,and KpnI restriction sites 503. The motif libraries can be limited tospecific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQsequences of Table 3. In this case, the motifs of the AE family are usedas the motif library, which are annealed to the 12-mer to create a“building block” length; e.g., a segment that encodes 36 amino acids.The gene encoding the XTEN sequence can be assembled by ligation andmultimerization of the “building blocks” until the desired length of theXTEN gene 504 is achieved. As illustrated in FIG. 32, the XTEN length inthis case is 48 amino acid residues, but longer lengths can be achievedby this process. For example, multimerization can be performed byligation, overlap extension, PCR assembly or similar cloning techniquesknown in the art. The XTEN gene can be cloned into a stuffer vector. Inthe example illustrated in FIG. 32, the vector can encode a Flagsequence 506 followed by a stuffer sequence that is flanked by BsaI,BbsI, and KpnI sites 507 and a CF gene (e.g., FVII) 508, resulting inthe gene encoding the CFXTEN 500, which, in this case encodes the fusionprotein in the configuration, N- to C-terminus, XTEN-FVII. As isapparent to one of ordinary skill in the art, the methods can be appliedto create constructs in alternative configurations and with varying XTENlengths.

DNA sequences encoding CF can be conveniently obtained by standardprocedures known in the art from a cDNA library prepared from anappropriate cellular source, from a genomic library, or may be createdsynthetically (e.g., automated nucleic acid synthesis) using DNAsequences obtained from publicly available databases, patents, orliterature references. A gene or polynucleotide encoding the CF portionof the protein or its complement can be then be cloned into a construct,such as those described herein, which can be a plasmid or other vectorunder control of appropriate transcription and translation sequences forhigh level protein expression in a biological system. A second gene orpolynucleotide coding for the XTEN portion or its complement (in thecase of FIG. 32 illustrated as an XTEN with 48 amino acid residues) canbe genetically fused to the nucleotides encoding the terminus of the CFgene by cloning it into the construct adjacent and in frame with thegene coding for the CF, through a ligation or multimerization step. Inthis manner, a chimeric DNA molecule coding for (or complementary to)the CFXTEN fusion protein Re generated within the construct. Optionally,a gene encoding for a second XTEN are inserted and ligated in-frame tothe nucleotides encoding the opposite terminus of the CFXTEN gene or canbe inserted within the CF-encoding region. The construct can be designedin different configurations to encode the various permutations of thefusion partners as a monomeric polypeptide. For example, the gene can becreated to encode the fusion protein in the order (N- to C-terminus):CF-XTEN; XTEN-CF; CF-XTEN-CF; XTEN-CF-XTEN; as well as multimers of theforegoing. Optionally, this chimeric DNA molecule is transferred orcloned into another construct that is a more appropriate expressionvector. At this point, a host cell capable of expressing the chimericDNA molecule is transformed with the chimeric DNA molecule. The vectorscontaining the DNA segments of interest can be transferred into anappropriate host cell by well-known methods, depending on the type ofcellular host, as described supra.

Host cells containing the XTEN-CF expression vector is cultured inconventional nutrient media modified as appropriate for activating thepromoter. The culture conditions, such as temperature, pH and the like,are those previously used with the host cell selected for expression,and will be apparent to the ordinarily skilled artisan. After expressionof the fusion protein, culture broth is harvested and separated from thecell mass and the resulting crude extract retained for purification ofthe fusion protein.

Gene expression are measured in a sample directly, for example, byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205(1980)], dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, gene expression is measured by immunological offluorescent methods, such as immunohistochemical staining of cells toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids may beeither monoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against the CF sequencepolypeptide using a synthetic peptide based on the sequences providedherein or against exogenous sequence fused to CF and encoding a specificantibody epitope. Examples of selectable markers are well known to oneof skill in the art and include reporters such as enhanced greenfluorescent protein (EGFP), beta-galactosidase (β-gal) orchloramphenicol acetyltransferase (CAT).

The CFXTEN polypeptide product is purified via methods known in the art.Procedures such as gel filtration, affinity purification, saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxyapatite adsorption chromatography, hydrophobicinteraction chromatography or gel electrophoresis are all techniquesthat may be used in the purification. Specific methods of purificationare described in Robert K. Scopes, Protein Purification: Principles andPractice, Charles R. Castor, ed., Springer-Verlag 1994, and Sambrook, etal., supra. Multi-step purification separations are also described inBaron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, etal., J. Chromatogr. A. 679:67-83 (1994).

As illustrated in FIG. 33, the isolated CFXTEN fusion proteins wouldthen be characterized for their chemical and activity properties.Isolated fusion protein is characterized, e.g., for sequence, purity,apparent molecular weight, solubility and stability using standardmethods known in the art. The fusion protein meeting expected standardswould then be evaluated for activity, which can be measured in vitro orin vivo by measuring one of the coagulation factor-associated parametersdescribed herein, using one or more assays disclosed herein, or usingthe assays of the Examples or Table 40.

In addition, the XTEN-CF fusion protein is administered to one or moreanimal species to determine standard pharmacokinetic parameters andpharmacodynamic properties, as described in Examples 30-33.

By the iterative process of producing, expressing, and recovering CFXTENconstructs, followed by their characterization using methods disclosedherein or others known in the art, the CFXTEN compositions comprising CFand an XTEN can be produced and evaluated by one of ordinary skill inthe art to confirm the expected properties such as enhanced solubility,enhanced stability, improved pharmacokinetics and reducedimmunogenicity, leading to an overall enhanced therapeutic activitycompared to the corresponding unfused CF. For those fusion proteins notpossessing the desired properties, a different sequence can beconstructed, expressed, isolated and evaluated by these methods in orderto obtain a composition with such properties.

Example 19: Construction of Expression Plasmids for FVII-XTEN

Construction of FVII-TEV-XTEN 864 Expression Vectors

The cloning vector containing the gene encoding FVII was purchased fromOriGene (SC109205). PCR reactions were performed to abolish BbsI andBsaI restriction sites within the FVII coding region. The resulting FVIIcoding region was then amplified using primers that introduced NheI andTEV-BsaI sequences on the 5′ and 3′ end respectively. The digested FVIIfragment was fused to BsaI/HindIII digested XTEN_AE864 fragment andinserted into NheI/HindIII digested pSecTag2C expression vector. Theligated DNA mixture was electroporated into XL1-Blue bacterial cells.Transformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The final construct is pCW0647.001which encodes the FVII-TEV-XTEN_AE864 protein (Table 23).

Construction of FVII-XTEN 864 Expression Vectors

FVII was amplified with pCW0647.001 as a template. The PCR primersintroduced NheI and BsaI restriction enzyme recognition sequences on the5′ and 3′ end respectively and deleted the TEV site. The NheI/BsaIdigested FVII fragment was fused to BsaI/HindIII digested XTEN_AE864fragment and inserted into NheI/HindIII digested pSecTag2C expressionvector. The ligated DNA mixture was electroporated into XL1-Bluebacterial cells. Transformants were screened by DNA miniprep and thedesired constructs were confirmed by DNA sequencing. The final constructis pCW0645.001 which encodes the FVII-XTEN_AE864 protein (Table 23).

Construction of Expression Vectors Encoding FVII-XTEN 864 Genes UsingMillipore Plasmids

Expression vector pCW0645.001 was digested with NheI and SalI. Theresulting 4091 bp fragment included nucleotides that encode theFVII-XTEN_AE864 protein. This fragment was ligated with NheI/SalIdigested CET1019-AS-puro, CET1019-HS-puro, SC AS-puro, or DC HS-puro(licensed from Millipore). These vectors feature a CMV promoter thatlies upstream of the gene insertion site, and the CET1019 vectors alsocontain a UCOE element upstream of the promoter. The ligated DNA mixturewas electroporated into XL1-Blue bacterial cells. Transformants werescreened by DNA miniprep and the desired constructs were confirmed byDNA sequencing. The resulting expression vectors were AC397 (pBC0013, SCAS puro-FVII-XTEN_AE864), AC402 (pBC0014, SC HS puro-FVII-XTEN_AE864),AC403 (pBC0015, CET1019 AS puro-FVII-XTEN_AE864), and AC404 (pBC0016,CET1019 HS puro-FVII-XTEN_AE864)

Construction of Expression Vectors Encoding FVII-XTEN 288 Genes

Expression vector pCW0645.001 was digested with BsaI and HindIII. Theresulting 6400 bp fragment was ligated with BsaI/HindIII digestedXTEN_AE288 fragment. The ligated DNA mixture was electroporated intoXL1-Blue bacterial cells. Transformants were screened by DNA miniprepand the desired constructs were confirmed by DNA sequencing. Theresulting expression vector was pBC0019 (pSecTag2C-FVII-XTEN_AE288).

Expression vector pBC0019 was digested with NheI and SalI. The resulting2363 bp fragment included nucleotides that encode the FVII-XTEN_AE288protein. This fragment was ligated with NheI/SalI digestedCET1019-AS-puro, or CET1019-HS-puro (licensed from Millipore). Thesevectors feature a CMV promoter and a UCOE element that lie upstream ofthe gene insertion site. The ligated DNA mixture was electroporated intoXL1-Blue bacterial cells. Transformants were screened by DNA miniprepand the desired constructs were confirmed by DNA sequencing. Theresulting expression vectors were AC405 (pBC0017, CET1019 ASpuro-FVII-XTEN_AE288), and AC398 (pBC0018, CET1019 HSpuro-FVII-XTEN_AE288) (Table 23).

TABLE 23 FVII amino acid and nucleic acid sequences SEQ SEQ Amino AcidID ID Name Sequence NO: Nucleic Acid Sequences NO: FVII-TEV-MVSQALRLLCLLLGLQ 596 ATGGTCTCCCAGGCCCTCAGGCTCCTCT 597 XTEN_AE864,GCLAAVFVTQEEAHGV GCCTTCTGCTTGGGCTTCAGGGCTGCCT pCW0647.001LHRRRRANAFLEELRP GGCTGCAGTGTTCGTAACCCAGGAGGAA GSLERECKEEQCSFEEGCCCACGGCGTCCTGCACCGGCGCCGGC AREIFKDAERTKLFWIGCGCCAACGCGTTCCTGGAGGAGCTACG SYSDGDQCASSPCQNGGCCGGGCTCCCTGGAGAGGGAGTGCAAG GSCKDQLQSYICFCLPGAGGAGCAGTGCTCCTTCGAGGAGGCCC AFEGRNCETHKDDQLIGGGAGATCTTCAAGGACGCGGAGAGGAC CVNENGGCEQYCSDHTGAAGCTGTTCTGGATTTCTTACAGTGAT GTKRSCRCHEGYSLLAGGGGACCAGTGTGCCTCAAGTCCATGCC DGVSCTPTVEYPCGKIAGAATGGGGGCTCCTGCAAGGACCAGCT PILEKRNASKPQGRIVCCAGTCCTATATCTGCTTCTGCCTCCCT GGKVCPKGECPWQVLLGCCTTCGAGGGCCGGAACTGTGAGACGC LVNGAQLCGGTLINTIACAAGGATGACCAGCTGATCTGTGTGAA WVVSAAHCFDKIKNWRCGAGAACGGCGGCTGTGAGCAGTACTGC NLIAVLGEHDLSEHDGAGTGACCACACGGGCACCAAGCGCTCCT DEQSRRVAQVIIPSTYGTCGGTGCCACGAGGGGTACTCTCTGCT VPGTTNHDIALLRLHQGGCAGACGGGGTGTCCTGCACACCCACA PVVLTDHVVPLCLPERGTTGAATATCCATGTGGAAAAATACCTA TFSERTLAFVRFSLVSTTCTAGAAAAAAGAAATGCCAGCAAACC GWGQLLDRGATALELMCCAAGGCCGAATTGTGGGGGGCAAGGTG VLNVPRLMTQDCLQQSTGCCCCAAAGGGGAGTGTCCATGGCAGG RKVGDSPNITEYMFCATCCTGTTGTTGGTGAATGGAGCTCAGTT GYSDGSKDSCKGDSGGGTGTGGGGGGACCCTGATCAACACCATC PHATHYRGTWYLTGIVTGGGTGGTCTCCGCGGCCCACTGTTTCG SWGQGCATVGHFGVYTACAAAATCAAGAACTGGAGGAACCTGAT RVSQYIEWLQKLMRSECGCGGTGCTGGGCGAGCACGACCTCAGC PRPGVLLRAPFPGPEGGAGCACGACGGGGATGAGCAGAGCCGGC PSENLYFQGGSPGSPAGGGTGGCGCAGGTCATCATCCCCAGCAC GSPTSTEEGTSESATPGTACGTCCCGGGCACCACCAACCACGAC ESGPGTSTEPSEGSAPATCGCGCTGCTCCGCCTGCACCAGCCCG GSPAGSPTSTEEGTSTTGGTCCTCACTGACCATGTGGTGCCCCT EPSEGSAPGTSIEPSECTGCCTGCCCGAACGGACGTTCTCTGAG GSAPGTSESATPESGPAGGACGCTGGCCTTCGTGCGCTTCTCAT GSEPATSGSETPGSEPTGGTCAGCGGCTGGGGCCAGCTGCTGGA ATSGSETPGSPAGSPTCCGTGGCGCCACGGCCCTGGAGCTCATG STEEGTSESATPESGPGTCCTCAACGTGCCCCGGCTGATGACCC GTSTEPSEGSAPGTSTAGGACTGCCTGCAGCAGTCACGGAAGGT EPSEGSAPGSPAGSPTGGGAGACTCCCCAAATATCACGGAGTAC STEEGTSTEPSEGSAPATGTTCTGTGCCGGCTACTCGGATGGCA GTSTEPSEGSAPGTSEGCAAGGACTCCTGCAAGGGGGACAGTGG SATPESGPGTSTEPSEAGGCCCACATGCCACCCACTACCGGGGC GSAPGTSESATPESGPACGTGGTACCTGACGGGCATCGTCAGCT GSEPATSGSETPGTSTGGGGCCAGGGCTGCGCAACCGTGGGCCA EPSEGSAPGTSTEPSECTTTGGGGTGTACACCAGGGTCTCCCAG GSAPGTSESATPESGPTACATCGAGTGGCTGCAAAAGCTCATGC GTSESATPESGPGSPAGCTCAGAGCCACGCCCAGGAGTCCTCCT GSPTSTEEGTSESATPGCGAGCCCCATTTCCCGGCCCAGAAGGC ESGPGSEPATSGSETPCCATCCGAAAATCTGTATTTTCAGGGTG GTSESATPESGPGTSTGGTCTCCAGGTTCTCCAGCCGGGTCCCC EPSEGSAPGTSTEPSEAACTTCGACCGAGGAAGGGACCTCCGAG GSAPGTSTEPSEGSAPTCAGCTACCCCGGAGTCCGGTCCTGGCA GTSTEPSEGSAPGTSTCCTCCACCGAACCATCGGAGGGCAGCGC EPSEGSAPGTSTEPSECCCTGGGAGCCCTGCCGGGAGCCCTACA GSAPGSPAGSPTSTEEAGCACCGAAGAGGGCACCAGTACAGAGC GTSTEPSEGSAPGTSECAAGTGAGGGGAGCGCCCCTGGTACTAG SATPESGPGSEPATSGTACTGAACCATCCGAGGGGTCAGCTCCA SETPGTSESATPESGPGGCACGAGTGAGTCCGCTACCCCCGAGA GSEPATSGSETPGTSEGCGGACCGGGCTCAGAGCCCGCCACGAG SATPESGPGTSTEPSETGGCAGTGAAACTCCAGGCTCAGAACCC GSAPGTSESATPESGPGCCACTAGTGGGTCAGAGACTCCAGGCA GSPAGSPTSTEEGSPAGCCCTGCCGGATCCCCTACGTCCACCGA GSPTSTEEGSPAGSPTGGAGGGAACATCTGAGTCCGCAACACCC STEEGTSESATPESGPGAATCCGGTCCAGGCACCTCCACGGAAC GTSTEPSEGSAPGTSECTAGTGAAGGCTCGGCACCAGGTACAAG SATPESGPGSEPATSGCACCGAACCTAGCGAGGGCAGCGCTCCC SETPGTSESATPESGPGGCAGCCCTGCCGGCAGCCCAACCTCAA GSEPATSGSETPGTSECTGAGGAGGGCACCAGTACTGAGCCCAG SATPESGPGTSTEPSECGAGGGATCAGCACCTGGCACCAGCACC GSAPGSPAGSPTSTEEGAACCTAGCGAGGGGAGCGCCCCTGGGA GTSESATPESGPGSEPCTAGCGAGTCAGCTACACCAGAGAGCGG ATSGSETPGTSESATPGCCTGGAACTTCTACCGAACCCAGTGAG ESGPGSPAGSPTSTEEGGATCCGCTCCAGGCACCTCCGAATCCG GSPAGSPTSTEEGTSTCAACCCCCGAATCCGGACCTGGCTCAGA EPSEGSAPGTSESATPGCCCGCCACCAGCGGGAGCGAAACCCCT ESGPGTSESATPESGPGGCACATCCACCGAGCCTAGCGAAGGGT GTSESATPESGPGSEPCCGCACCCGGCACCAGTACAGAGCCTAG ATSGSETPGSEPATSGCGAGGGATCAGCACCTGGCACCAGTGAA SETPGSPAGSPTSTEETCTGCTACACCAGAGAGCGGCCCTGGAA GTSTEPSEGSAPGTSTCCTCCGAGTCCGCTACCCCCGAGAGCGG EPSEGSAPGSEPATSGGCCAGGTTCTCCTGCTGGCTCCCCCACC SETPGTSESATPESGPTCAACAGAAGAGGGGACAAGCGAAAGCG GTSTEPSEGSAPGSSSCTACGCCTGAGAGTGGCCCTGGCTCTGA GCCAGCCACCTCCGGCTCTGAAACCCCTGGCACTAGTGAGTCTGCCACGCCTGAGT CCGGACCCGGGACCTCTACTGAGCCCTCGGAGGGGAGCGCTCCTGGCACGAGTACA GAACCTTCCGAAGGAAGTGCACCGGGCACAAGCACCGAGCCTTCCGAAGGCTCTGC TCCCGGAACCTCTACCGAACCCTCTGAAGGGTCTGCACCCGGCACGAGCACCGAAC CCAGCGAAGGGTCAGCGCCTGGGACCTCAACAGAGCCCTCGGAAGGATCAGCGCCT GGAAGCCCTGCAGGGAGTCCAACTTCCACGGAAGAAGGAACGTCTACAGAGCCATC AGAGGGGTCCGCACCAGGTACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGGGT CCGAACCTGCCACCTCCGGCTCTGAAACTCCAGGGACCTCCGAATCTGCCACACCC GAGAGCGGCCCTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACCTGGCACCTC CGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAGCACCGAGCCATCCGAGGGAT CCGCCCCAGGCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCGCT GGCTCCCCCACCAGTACCGAGGAGGGGTCCCCCGCTGGAAGTCCAACAAGCACTGA GGAAGGGTCCCCTGCCGGCTCCCCCACAAGTACCGAAGAGGGCACAAGTGAGAGCG CCACTCCCGAGTCCGGGCCTGGCACCAGCACAGAGCCTTCCGAGGGGTCCGCACCA GGTACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTC CGGGTCTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGT CCGAGCCCGCCACTTCCGGCTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCA GAGTCAGGACCAGGAACATCTACAGAGCCCTCTGAAGGCTCCGCTCCAGGGTCCCC AGCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCACACCCGAAT CAGGGCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAG TCCGCCACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGA GGAGGGATCTCCTGCTGGCTCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGC CATCCGAGGGATCAGCTCCCGGCACCTCAGAGTCGGCAACCCCGGAGTCTGGACCC GGAACTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGAATCAGCAAC ACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGAT CAGAACCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACC TCCACTGAGGAGGGCACCAGCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAG CACTGAACCCAGTGAGGGTTCAGCACCCGGCTCTGAGCCGGCCACAAGTGGCAGTG AGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGCACTAGTACC GAGCCCTCTGAAGGCAGTGCGCCAGGTT CGTCTTCATAAFVII- MVSQALRLLCLLLGLQ 598 ATGGTCTCCCAGGCCCTCAGGCTCCTCT 599 XTEN_AE864,GCLAAVFVTQEEAHGV GCCTTCTGCTTGGGCTTCAGGGCTGCCT pCW0645.001LHRRRRANAFLEELRP GGCTGCAGTGTTCGTAACCCAGGAGGAA GSLERECKEEQCSFEEGCCCACGGCGTCCTGCACCGGCGCCGGC AREIFKDAERTKLFWIGCGCCAACGCGTTCCTGGAGGAGCTACG SYSDGDQCASSPCQNGGCCGGGCTCCCTGGAGAGGGAGTGCAAG GSCKDQLQSYICFCLPGAGGAGCAGTGCTCCTTCGAGGAGGCCC AFEGRNCETHKDDQLIGGGAGATCTTCAAGGACGCGGAGAGGAC CVNENGGCEQYCSDHTGAAGCTGTTCTGGATTTCTTACAGTGAT GTKRSCRCHEGYSLLAGGGGACCAGTGTGCCTCAAGTCCATGCC DGVSCTPTVEYPCGKIAGAATGGGGGCTCCTGCAAGGACCAGCT PILEKRNASKPQGRIVCCAGTCCTATATCTGCTTCTGCCTCCCT GGKVCPKGECPWQVLLGCCTTCGAGGGCCGGAACTGTGAGACGC LVNGAQLCGGTLINTIACAAGGATGACCAGCTGATCTGTGTGAA WVVSAAHCFDKIKNWRCGAGAACGGCGGCTGTGAGCAGTACTGC NLIAVLGEHDLSEHDGAGTGACCACACGGGCACCAAGCGCTCCT DEQSRRVAQVIIPSTYGTCGGTGCCACGAGGGGTACTCTCTGCT VPGTTNHDIALLRLHQGGCAGACGGGGTGTCCTGCACACCCACA PVVLTDHVVPLCLPERGTTGAATATCCATGTGGAAAAATACCTA TFSERTLAFVRFSLVSTTCTAGAAAAAAGAAATGCCAGCAAACC GWGQLLDRGATALELMCCAAGGCCGAATTGTGGGGGGCAAGGTG VLNVPRLMTQDCLQQSTGCCCCAAAGGGGAGTGTCCATGGCAGG RKVGDSPNITEYMFCATCCTGTTGTTGGTGAATGGAGCTCAGTT GYSDGSKDSCKGDSGGGTGTGGGGGGACCCGATCAACACCATCT PHATHYRGTWYLTGIVGGGTGGTCTCCGCGGCCCACTGTTTCGA SWGQGCATVGHFGVYTCAAAATCAAGAACTGGAGGAACCTGATC RVSQYIEWLQKLMRSEGCGGTGCTGGGCGAGCACGACCTCAGCG PRPGVLLRAPFPGSPGAGCACGACGGGGATGAGCAGAGCCGGCG SPAGSPTSTEEGTSESGGTGGCGCAGGTCATCATCCCCAGCACG ATPESGPGTSTEPSEGTACGTCCCGGGCACCACCAACCACGACA SAPGSPAGSPTSTEEGTCGCGCTGCTCCGCCTGCACCAGCCCGT TSTEPSEGSAPGTSTEGGTCCTCACTGACCATGTGGTGCCCCTC PSEGSAPGTSESATPETGCCTGCCCGAACGGACGTTCTCTGAGA SGPGSEPATSGSETPGGGACGCTGGCCTTCGTGCGCTTCTCATT SEPATSGSETPGSPAGGGTCAGCGGCTGGGGCCAGCTGCTGGAC SPTSlEEGTSESATPECGTGGCGCCACGGCCCTGGAGCTCATGG SGPGTSTEPSEGSAPGTCCTCAACGTGCCCCGGCTGATGACCCA TSTEPSEGSAPGSPAGGGACTGCCTGCAGCAGTCACGGAAGGTG SPTSlEEGTSTEPSEGGGAGACTCCCCAAATATCACGGAGTACA SAPGTSTEPSEGSAPGTGTTCTGTGCCGGCTACTCGGATGGCAG TSESATPESGPGTSTECAAGGACTCCTGCAAGGGGGACAGTGGA PSEGSAPGTSESATPEGGCCCACATGCCACCCACTACCGGGGCA SGPGSEPATSGSETPGCGTGGTACCTGACGGGCATCGTCAGCTG TSTEPSEGSAPGTSTEGGGCCAGGGCTGCGCAACCGTGGGCCAC PSEGSAPGTSESATPETTTGGGGTGTACACCAGGGTCTCCCAGT SGPGTSESATPESGPGACATCGAGTGGCTGCAAAAGCTCATGCG SPAGSPTSlEEGTSESCTCAGAGCCACGCCCAGGAGTCCTCCTG ATPESGPGSEPATSGSCGAGCCCCATTTCCCGGAGGTAGCCCGG ETPGTSESATPESGPGCTGGCTCTCCTACCTCTACTGAGGAAGG TSTEPSEGSAPGTSTETACTTCTGAAAGCGCTACTCCTGAGTCT PSEGSAPGTSTEPSEGGGTCCAGGTACCTCTACTGAACCGTCCG SAPGTSTEPSEGSAPGAAGGTAGCGCTCCAGGTAGCCCAGCAGG TSTEPSEGSAPGTSTECTCTCCGACTTCCACTGAGGAAGGTACT PSEGSAPGSPAGSPTSTCTACTGAACCTTCCGAAGGCAGCGCAC TEEGTSTEPSEGSAPGCAGGTACCTCTACTGAACCTTCTGAGGG TSESATPESGPGSEPACAGCGCTCCAGGTACTTCTGAAAGCGCT TSGSETPGTSESATPEACCCCGGAATCTGGCCCAGGTAGCGAAC SGPGSEPATSGSETPGCGGCTACTTCTGGTTCTGAAACCCCAGG TSESATPESGPGTSTETAGCGAACCGGCTACCTCCGGTTCTGAA PSEGSAPGTSESATPEACTCCAGGTAGCCCGGCAGGCTCTCCGA SGPGSPAGSPTSFEEGCCTCTACTGAGGAAGGTACTTCTGAAAG SPAGSPTSlEEGSPAGCGCAACCCCGGAGTCCGGCCCAGGTACC SPTSTEEGTSESATPETCTACCGAACCGTCTGAGGGCAGCGCAC SGPGTSTEPSEGSAPGCAGGTACTTCTACCGAACCGTCCGAGGG TSESATPESGPGSEPATAGCGCACCAGGTAGCCCAGCAGGTTCT TSGSETPGTSESATPECCTACCTCCACCGAGGAAGGTACTTCTA SGPGSEPATSGSETPGCCGAACCGTCCGAGGGTAGCGCACCAGG TSESATPESGPGTSTETACCTCTACTGAACCTTCTGAGGGCAGC PSEGSAPGSPAGSPTSGCTCCAGGTACTTCTGAAAGCGCTACCC TEEGTSESATPESGPGCGGAGTCCGGTCCAGGTACTTCTACTGA SEPATSGSETPGTSESACCGTCCGAAGGTAGCGCACCAGGTACT ATPESGPGSPAGSPTSTCTGAAAGCGCAACCCCTGAATCCGGTC TEEGSPAGSPTSTEEGCAGGTAGCGAACCGGCTACTTCTGGCTC TSTEPSEGSAPGTSESTGAGACTCCAGGTACTTCTACCGAACCG ATPESGPGTSESATPETCCGAAGGTAGCGCACCAGGTACTTCTA SGPGTSESATPESGPGCTGAACCGTCTGAAGGTAGCGCACCAGG SEPATSGSETPGSEPATACTTCTGAAAGCGCAACCCCGGAATCC TSGSETPGSPAGSPTSGGCCCAGGTACCTCTGAAAGCGCAACCC TEEGTSTEPSEGSAPGCGGAGTCCGGCCCAGGTAGCCCTGCTGG TSTEPSEGSAPGSEPACTCTCCAACCTCCACCGAAGAAGGTACC TSGSETPGTSESATPETCTGAAAGCGCAACCCCTGAATCCGGCC SGPGTSTEPSEGSAPGCAGGTAGCGAACCGGCAACCTCCGGTTC SSS TGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTA CTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGC GCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGA ACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCAC CAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCT CCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG TACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCG GCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC GAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGA ATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTG AAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACC GAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGG CTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCC CAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCA ACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG TACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTG GCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACT TCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTC CACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAAC CGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC GGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGG CTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCAC CAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCT ACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGG TAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCG GTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACT TCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGG CAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG AAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCAGGTTAA FVII- MVSQALRLLCLLLGLQ 600ATGGTGTCCCAGGCCCTCAGGCTCCTCT 601 XTEN_AE288, GCLAAVFVTQEEAHGVGCCTTCTGCTTGGGCTTCAGGGCTGCCT pBC0019 LHRRRRANAFLEELRPGGCTGCAGTGTTCGTAACCCAGGAGGAA GSLERECKEEQCSFEEGCCCACGGCGTCCTGCACCGGCGCCGGC AREIFKDAERTKLFWIGCGCCAACGCGTTCCTGGAGGAGCTACG SYSDGDQCASSPCQNGGCCGGGCTCCCTGGAGAGGGAGTGCAAG GSCKDQLQSYICFCLPGAGGAGCAGTGCTCCTTCGAGGAGGCCC AFEGRNCETHKDDQLIGGGAGATCTTCAAGGACGCGGAGAGGAC CVNENGGCEQYCSDHTGAAGCTGTTCTGGATTTCTTACAGTGAT GTKRSCRCHEGYSLLAGGGGACCAGTGTGCCTCAAGTCCATGCC DGVSCTPTVEYPCGKIAGAATGGGGGCTCCTGCAAGGACCAGCT PILEKRNASKPQGRIVCCAGTCCTATATCTGCTTCTGCCTCCCT GGKVCPKGECPWQVLLGCCTTCGAGGGCCGGAACTGTGAGACGC LVNGAQLCGGTLINTIACAAGGATGACCAGCTGATCTGTGTGAA WVVSAAHCFDKIKNWRCGAGAACGGCGGCTGTGAGCAGTACTGC NLIAVLGEHDLSEHDGAGTGACCACACGGGCACCAAGCGCTCCT DEQSRRVAQVIIPSTYGTCGGTGCCACGAGGGGTACTCTCTGCT VPGTTNHDIALLRLHQGGCAGACGGGGTGTCCTGCACACCCACA PVVLTDHVVPLCLPERGTTGAATATCCATGTGGAAAAATACCTA TFSERTLAFVRFSLVSTTCTAGAAAAAAGAAATGCCAGCAAACC GWGQLLDRGATALELMCCAAGGCCGAATTGTGGGGGGCAAGGTG VLNVPRLMTQDCLQQSTGCCCCAAAGGGGAGTGTCCATGGCAGG RKVGDSPNITEYMFCATCCTGTTGTTGGTGAATGGAGCTCAGTT GYSDGSKDSCKGDSGGGTGTGGGGGGACCCTGATCAACACCATC PHATHYRGTWYLTGIVTGGGTGGTGTCCGCGGCCCACTGTTTCG SWGQGCATVGHFGVYTACAAAATCAAGAACTGGAGAACCTGATC RVSQYIEWLQKLMRSEGCGGTGCTGGGCGAGCACGACCTCAGCG PRPGVLLRAPFPGSPGAGCACGACGGGGATGAGCAGAGCCGGCG TSESATPESGPGSEPAGGTGGCGCAGGTCATCATCCCCAGCACG TSGSETPGTSESATPETACGTCCCGGGCACCACCAACCACGACA SGPGSEPATSGSETPGTCGCGCTGCTCCGCCTGCACCAGCCCGT TSESATPESGPGTSTEGGTCCTCACTGACCATGTGGTGCCCCTC PSEGSAPGSPAGSPTSTGCCTGCCCGAACGGACGTTCTCTGAGA TEEGTSESATPESGPGGGACGCTGGCCTTCGTGCGCTTCTCATT SEPATSGSETPGTSESGGTCAGCGGCTGGGGCCAGCTGCTGGAC ATPESGPGSPAGSPTSCGTGGCGCCACGGCCCTGGAGCTCATGG TEEGSPAGSPTSTEEGTCCTCAACGTGCCCCGGCTGATGACCCA TSTEPSEGSAPGTSESGGACTGCCTGCAGCAGTCACGGAAGGTG ATPESGPGTSESATPEGGAGACTCCCCAAATATCACGGAGTACA SGPGTSESATPESGPGTGTTCTGTGCCGGCTACTCGGATGGCAG SEPATSGSETPGSEPACAAGGACTCCTGCAAGGGGGACAGTGGA TSGSETPGSPAGSPTSGGCCCACATGCCACCCACTACCGGGGCA TEEGTSTEPSEGSAPGCGTGGTACCTGACGGGCATCGTCAGCTG TSTEPSEGSAPGSEPAGGGCCAGGGCTGCGCAACCGTGGGCCAC TSGSETPGTSESATPETTTGGGGTGTACACCAGGGTGTCCCAGT SGPGTSTEPSEGSAPGACATCGAGTGGCTGCAAAAGCTCATGCG SSS CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTTCCCGGGTCTCCAGGTA CCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTCCGGG TCTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGA GCCCGCCACTTCCGGCTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCAGAGT CAGGACCAGGAACATCTACAGAGCCCTCTGAAGGCTCCGCTCCAGGGTCCCCAGCC GGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCACACCCGAATCAGG GCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCG CCACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGAGGAG GGATCTCCTGCTGGCTCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGCCATC CGAGGGATCAGCTCCCGGCACCTCAGAGTCGGCAACCCCGGAGTCTGGACCCGGAA CTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGAATCAGCAACACCC GAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGATCAGA ACCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCA CTGAGGAGGGCACCAGCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAGCACT GAACCCAGTGAGGGTTCAGCACCCGGCTCTGAGCCGGCCACAAGTGGCAGTGAGAC ACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGCACTAGTACCGAGC CCTCTGAAGGCAGTGCGCCAGGTTCGTC TTCATAA

Example 20: Construction of Expression Plasmids for FIX-XTEN

Construction of FIX-XTEN 864 Genes and Vectors

The cloning vector containing the gene encoding FIX was purchased fromOriGene (SC126517). PCR reactions were performed to abolish two BbsIrestriction sites within the FIX coding region. The resulting FIX codingregion was then amplified using primers that introduced NheI and BsaIrestriction enzyme recognition sequences on the 5′ and 3′ endrespectively. The digested FIX fragment was fused to BsaI/HindIIIdigested XTEN_AM864, AE864, AF864, or AG864 fragments and inserted intoNheI/HindIII digested pSecTag2C expression vector. The final constructsare AC282 (pCW0562, FIX-XTEN_AM864), AC283 (pCW0563, FIX-XTEN_AE864),pCW0564 (FIX-XTEN_AF864), and pCW0565 (FIX-XTEN_AG864) (Table 24).

Construction of Expression Vectors for FIX Helper Genes

The cloning vector containing the gene encoding PC5 was purchased fromOriGene (SC310051). The PC5 coding region was amplified using primersthat introduced NotI and BstBI restriction enzyme recognition sequences.The NotI/BstBI digested PC5 fragment was ligated with NotI/BstBIdigested CET1019-HD-puro or DC-HD-puro vectors. Both CET1019-HD-puro andDC-HD-puro feature dual cassettes where a CMV promoter lies upstream ofthe gene insertion site, CET1019-HD-puro also contains a UCOE elementupstream of the promoter. The ligated DNA mixture was electroporatedinto XL1-Blue bacterial cells. Transformants were screened by DNAminiprep and the desired constructs were confirmed by DNA sequencing.The resulting expression vectors are pBC0037 (DC-HD-puro-PC5) andpBC0038 (CET1019 I-ID-puro-PC5).

Construction of FIX-XTEN and PC5 Dual Expression Vectors

The pBC0037 and pBC0038 constructs were digested with NheI and SalI andligated with the NheI/SalI digested FIX-XTEN_AE864. The ligated DNAmixture was electroporated into XL1-Blue bacterial cells. Transformantswere screened by DNA miniprep and the desired constructs were confirmedby DNA sequencing. The resulting expression vectors were pBC0035(DC-HD-puro-FIX-XTEN_AE864-PC5) and pBC0036(CET1019-HD-puro-FIX-XTEN_AE864-PC5).

TABLE 24 FIX amino acid and nucleic acid sequences SEQ SEQ Amino Acid IDID Name Sequence NO: Nucleic Acid Sequences NO: FIX- MQRVNMIMAESPGLITI602 ATGCAGCGCGTGAACATGATCAT 603 XTEN_AM864, CLLGYLLSAECTVFLDHGGCAGAATCACCAGGCCTCATCA pCW0562 ENANKILNRPKRYNSGKCCATCTGCCTTTTAGGATATCTA LEEFVQGNLERECMEEK CTCAGTGCTGAATGTACAGTTTTCSFEEAREVFENTERTT TCTTGATCATGAAAACGCCAACA EFWKQYVDGDQCESNPCAAATTCTGAATCGGCCAAAGAGG LNGGSCKDDINSYECWC TATAATTCAGGTAAATTGGAAGAPFGFEGKNCELDVTCNI GTTTGTTCAAGGGAACCTTGAGA KNGRCEQFCKNSADNKVGAGAATGTATGGAAGAAAAGTGT VCSCTEGYRLAENQKSC AGTTTTGAAGAAGCACGAGAAGTEPAVPFPCGRVSVSQTS TTTTGAAAACACTGAAAGAACAA KLTRAETVFPDVDYVNSCTGAATTTTGGAAGCAGTATGTT TEAETILDNITQSTQSF GATGGAGATCAGTGTGAGTCCAANDFTRVVGGEDAKPGQF TCCATGTTTAAATGGCGGCAGTT PWQVVLNGKVDAFCGGSGCAAGGATGACATTAATTCCTAT IVNEKWIVTAAHCVETG GAATGTTGGTGTCCCTTTGGATTVKITVVAGEHNIEETEH TGAAGGAAAGAACTGTGAATTAG TEQKRNVIRIIPHHNYNATGTAACATGTAACATTAAGAAT AAINKYNHDIALLELDE GGCAGATGCGAGCAGTTTTGTAAPLVLNSYVTPICIADKE AAATAGTGCTGATAACAAGGTGG YTNIFLKFGSGYVSGWGTTTGCTCCTGTACTGAGGGATAT RVFHKGRSALVLQYLRV CGACTTGCAGAAAACCAGAAGTCPLVDRATCLRSTKFTIY CTGTGAACCAGCAGTGCCATTTC NNMFCAGFHEGGRDSCQCATGTGGAAGAGTTTCTGTTTCA GDSGGPHVTEVEGTSFL CAAACTTCTAAGCTCACCCGTGCTGIISWGEECAMKGKYG TGAGACTGTTTTTCCTGATGTGG IYTKVSRYVNWIKEKTKACTATGTAAATTCTACTGAAGCT LTGGTSTEPSEGSAPGS GAAACCATTTTGGATAACATCACEPATSGSETPGSPAGSP TCAAAGCACCCAATCATTTAATG TSTEEGSTSSTAESPGPACTTCACTCGGGTTGTTGGTGGA GTSTPESGSASPGSTSE GAAGATGCCAAACCAGGTCAATTSPSGTAPGSTSESPSGT CCCTTGGCAGGTTGTTTTGAATG APGTSTPESGSASPGTSGTAAAGTTGATGCATTCTGTGGA TPESGSASPGSEPATSG GGCTCTATCGTTAATGAAAAATGSETPGTSESATPESGPG GATTGTAACTGCTGCCCACTGTG SPAGSPTSTEEGTSTEPTTGAAACTGGTGTTAAAATTACA SEGSAPGTSESATPESG GTTGTCGCAGGTGAACATAATATPGTSTEPSEGSAPGTST TGAGGAGACAGAACATACAGAGC EPSEGSAPGSPAGSPTSAAAAGCGAAATGTGATTCGAATT TEEGTSIEPSEGSAPGT ATTCCTCACCACAACTACAATGCSTEPSEGSAPGTSESAT AGCTATTAATAAGTACAACCATG PESGPGTSESATPESGPACATTGCCCTTCTGGAACTGGAC GTSTEPSEGSAPGTSTE GAACCCTTAGTGCTAAACAGCTAPSEGSAPGTSESATPES CGTTACACCTATTTGCATTGCTG GPGTSTEPSEGSAPGSEACAAGGAATACACGAACATCTTC PATSGSETPGSPAGSPT CTCAAATTTGGATCTGGCTATGTSTEEGSSTPSGATGSPG AAGTGGCTGGGGAAGAGTCTTCC TPGSGTASSSPGSSTPSACAAAGGGAGATCAGCTTTAGTT GATGSPGTSTEPSEGSA CTTCAGTACCTTAGAGTTCCACTPGTSTEPSEGSAPGSEP TGTTGACCGAGCCACATGTCTTC ATSGSETPGSPAGSPTSGATCTACAAAGTTCACCATCTAT TEEGSPAGSPTSTEEGT AACAACATGTTCTGTGCTGGCTTSTEPSEGSAPGASASGA CCATGAAGGAGGTAGAGATTCAT PSTGGTSESATPESGPGGTCAAGGAGATAGTGGGGGACCC SPAGSPTSTEEGSPAGS CATGTTACTGAAGTGGAAGGGACPTSTEEGSTSSTAESPG CAGTTTCTTAACTGGAATTATTA PGSTSESPSGTAPGTSPGCTGGGGTGAAGAGTGTGCAATG SGESSTAPGTPGSGTAS AAAGGCAAATATGGAATATATACSSPGSSTPSGATGSPGS CAAGGTATCCCGGTATGTCAACT SPSASTGTGPGSEPATSGGATTAAGGAAAAAACAAAGCTC GSETPGTSESATPESGP ACTGGAGGTACTTCTACTGAACCGSEPATSGSETPGSTSS GTCTGAAGGCAGCGCACCAGGTA TAESPGPGSTSSTAESPGCGAACCGGCTACTTCCGGTTCT GPGTSPSGESSTAPGSE GAAACCCCAGGTAGCCCAGCAGGPATSGSETPGSEPATSG TTCTCCAACTTCTACTGAAGAAG SETPGTSTEPSEGSAPGGTTCTACCAGCTCTACCGCAGAA STSSTAESPGPGTSTPE TCTCCTGGTCCAGGTACCTCTACSGSASPGSTSESPSGTA TCCGGAAAGCGGCTCTGCATCTC PGTSTEPSEGSAPGTSTCAGGTTCTACTAGCGAATCTCCT EPSEGSAPGTSTEPSEG TCTGGCACTGCACCAGGTTCTACSAPGSSTPSGATGSPGS TAGCGAATCCCCGTCTGGTACTG SPSASTGTGPGASPGTSCTCCAGGTACTTCTACTCCTGAA STGSPGSEPATSGSETP AGCGGTTCCGCTTCTCCAGGTACGTSESATPESGPGSPAG CTCTACTCCGGAAAGCGGTTCTG SPTSTEEGSSTPSGATGCATCTCCAGGTAGCGAACCGGCA SPGSSPSASTGTGPGAS ACCTCCGGCTCTGAAACCCCAGGPGTSSTGSPGTSESATP TACCTCTGAAAGCGCTACTCCTG ESGPGTSTEPSEGSAPGAATCCGGCCCAGGTAGCCCGGCA TSTEPSEGSAPG GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTG AGGGCAGCGCTCCAGGTACTTCT GAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGT CCGAAGGTAGCGCACCAGGTACT TCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTT CTCCTACCTCCACCGAGGAAGGT ACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCG AACCTTCCGAGGGCAGCGCACCA GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTG AAAGCGCTACTCCTGAATCCGGT CCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCT CTACCGAACCGTCCGAGGGCAGC GCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTA CTTCTACTGAACCTTCCGAAGGT AGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAG GTAGCCCGGCTGGCTCTCCGACC TCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTC CAGGTACTCCGGGCAGCGGTACT GCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCT CTCCAGGTACCTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTA GCGCTCCAGGTAGCGAACCGGCA ACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTT CTACTGAGGAAGGTAGCCCGGCT GGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCG AAGGTAGCGCTCCAGGTGCAAGC GCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTC CTGAGTCCGGCCCAGGTAGCCCG GCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTC CAACTTCTACTGAAGAAGGTTCT ACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAAT CTCCGTCTGGCACCGCACCAGGT ACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCA GCGGTACCGCTTCTTCCTCTCCA GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCC CGTCTGCATCTACCGGTACCGGC CCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTT CTGAAAGCGCTACTCCGGAATCC GGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTT CCACCAGCTCTACTGCAGAATCT CCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG GTACTTCTCCTAGCGGCGAATCT TCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTC CAGGTAGCGAACCTGCAACCTCC GGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCG CACCAGGTTCTACCAGCTCTACC GCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTG CATCTCCAGGTTCTACTAGCGAA TCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAG GCAGCGCTCCAGGTACCTCTACT GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTG AAGGTAGCGCACCAGGTAGCTCT ACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTT CCACTGGTACTGGCCCAGGTGCT TCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTA CCTCCGGTTCTGAAACCCCAGGT ACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAG GTTCTCCTACCTCCACTGAGGAA GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCC CGTCTGCTTCCACTGGTACTGGC CCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCT CTGAAAGCGCTACTCCGGAGTCT GGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTA CTTCTACTGAACCGTCCGAAGGT AGCGCACCAGGTTAA FIX-MQRVNMIMAESPGLITI 604 ATGCAGCGCGTGAACATGATCAT 605 XTEN_AE864,CLLGYLLSAECTVFLDH GGCAGAATCACCAGGCCTCATCA  pCW0563 ENANKILNRPKRYNSGKCCATCTGCCTTTTAGGATATCTA  LEEFVQGNLERECMEEK CTCAGTGCTGAATGTACAGTTTT CSFEEAREVFENTERTT TCTTGATCATGAAAACGCCAACA  EFWKQYVDGDQCESNPCAAATTCTGAATCGGCCAAAGAGG  LNGGSCKDDINSYECWC TATAATTCAGGTAAATTGGAAGA PFGFEGKNCELDVTCNI GTTTGTTCAAGGGAACCTTGAGA  KNGRCEQFCKNSADNKVGAGAATGTATGGAAGAAAAGTGT  VCSCTEGYRLAENQKSC AGTTTTGAAGAAGCACGAGAAGT EPAVPFPCGRVSVSQTS TTTTGAAAACACTGAAAGAACAA  KLTRAETVFPDVDYVNSCTGAATTTTGGAAGCAGTATGTT  TEAETILDNITQSTQSF GATGGAGATCAGTGTGAGTCCAA NDFTRVVGGEDAKPGQF TCCATGTTTAAATGGCGGCAGTT  PWQVVLNGKVDAFCGGSGCAAGGATGACATTAATTCCTAT  IVNEKWIVTAAHCVETG GAATGTTGGTGTCCCTTTGGATT VKITVVAGEHNIEETEH TGAAGGAAAGAACTGTGAATTAG  TEQKRNVIRIIPHHNYNATGTAACATGTAACATTAAGAAT  AAINKYNHDIALLELDE GGCAGATGCGAGCAGTTTTGTAA PLVLNSYVTPICIADKE AAATAGTGCTGATAACAAGGTGG  YTNIFLKFGSGYVSGWGTTTGCTCCTGTACTGAGGGATAT  RVFHKGRSALVLQYLRV CGACTTGCAGAAAACCAGAAGTC PLVDRATCLRSTKFTIY CTGTGAACCAGCAGTGCCATTTC  NNMFCAGFHEGGRDSCQCATGTGGAAGAGTTTCTGTTTCA  GDSGGPHVTEVEGTSFL CAAACTTCTAAGCTCACCCGTGC TGIISWGEECAMKGKYG TGAGACTGTTTTTCCTGATGTGG  IYTKVSRYVNWIKEKTKACTATGTAAATTCTACTGAAGCT  LTGGSPAGSPTSTEEGT GAAACCATTTTGGATAACATCAC SESATPESGPGTSTEPS TCAAAGCACCCAATCATTTAATG  EGSAPGSPAGSPTSTEEACTTCACTCGGGTTGTTGGTGGA  GTSTEPSEGSAPGTSTE GAAGATGCCAAACCAGGTCAATT PSEGSAPGTSESATPES CCCTTGGCAGGTTGTTTTGAATG  GPGSEPATSGSETPGSEGTAAAGTTGATGCATTCTGTGGA  PATSGSETPGSPAGSPT GGCTCTATCGTTAATGAAAAATG STEEGTSESATPESGPG GATTGTAACTGCTGCCCACTGTG  TSTEPSEGSAPGTSTEPTTGAAACTGGTGTTAAAATTACA  SEGSAPGSPAGSPTSTE GTTGTCGCAGGTGAACATAATAT EGTSTEPSEGSAPGTST TGAGGAGACAGAACATACAGAGC  EPSEGSAPGTSESATPEAAAAGCGAAATGTGATTCGAATT  SGPGTSTEPSEGSAPGT ATTCCTCACCACAACTACAATGC SESATPESGPGSEPATS AGCTATTAATAAGTACAACCATG  GSETPGTSTEPSEGSAPACATTGCCCTTCTGGAACTGGAC  GTSTEPSEGSAPGTSES GAACCCTTAGTGCTAAACAGCTA ATPESGPGTSESATPES CGTTACACCTATTTGCATTGCTG  GPGSPAGSPTSTEEGTSACAAGGAATACACGAACATCTTC  ESATPESGPGSEPATSG CTCAAATTTGGATCTGGCTATGT SETPGTSESATPESGPG AAGTGGCTGGGGAAGAGTCTTCC  TSTEPSEGSAPGTSTEPACAAAGGGAGATCAGCTTTAGTT  SEGSAPGTSTEPSEGSA CTTCAGTACCTTAGAGTTCCACT PGTSTEPSEGSAPGTST TGTTGACCGAGCCACATGTCTTC  EPSEGSAPGTSTEPSEGGATCTACAAAGTTCACCATCTAT  SAPGSPAGSPTSTEEGT AACAACATGTTCTGTGCTGGCTT STEPSEGSAPGTSESAT CCATGAAGGAGGTAGAGATTCAT  PESGPGSEPATSGSETPGTCAAGGAGATAGTGGGGGACCC  GTSESATPESGPGSEPA CATGTTACTGAAGTGGAAGGGAC TSGSETPGTSESATPES CAGTTTCTTAACTGGAATTATTA  GPGTSTEPSEGSAPGTSGCTGGGGTGAAGAGTGTGCAATG  ESATPESGPGSPAGSPT AAAGGCAAATATGGAATATATAC STEEGSPAGSPTSTEEG CAAGGTATCCCGGTATGTCAACT  SPAGSPTSTEEGTSESAGGATTAAGGAAAAAACAAAGCTC  TPESGPGTSTEPSEGSA ACTGGAGGTAGCCCGGCTGGCTC PGTSESATPESGPGSEP TCCTACCTCTACTGAGGAAGGTA  ATSGSETPGTSESATPECTTCTGAAAGCGCTACTCCTGAG  SGPGSEPATSGSETPGT TCTGGTCCAGGTACCTCTACTGA SESATPESGPGTSTEPS ACCGTCCGAAGGTAGCGCTCCAG  EGSAPGSPAGSPTSTEEGTAGCCCAGCAGGCTCTCCGACT  GTSESATPESGPGSEPA TCCACTGAGGAAGGTACTTCTAC TSGSETPGTSESATPES TGAACCTTCCGAAGGCAGCGCAC  GPGSPAGSPTSTEEGSPCAGGTACCTCTACTGAACCTTCT  AGSPTSTEEGTSTEPSE GAGGGCAGCGCTCCAGGTACTTC GSAPGTSESATPESGPG TGAAAGCGCTACCCCGGAATCTG  TSESATPESGPGTSESAGCCCAGGTAGCGAACCGGCTACT  TPESGPGSEPATSGSET TCTGGTTCTGAAACCCCAGGTAG PGSEPATSGSETPGSPA CGAACCGGCTACCTCCGGTTCTG  GSPTSTEEGTSTEPSEGAAACTCCAGGTAGCCCGGCAGGC  SAPGTSTEPSEGSAPGS TCTCCGACCTCTACTGAGGAAGGEPATSGSETPGTSESAT TACTTCTGAAAGCGCAACCCCGG PESGPGTSTEPSEGSAPAGTCCGGCCCAGGTACCTCTACC G GAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCG AGGGTAGCGCACCAGGTAGCCCA GCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGT CCGAGGGTAGCGCACCAGGTACC TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCG CTACCCCGGAGTCCGGTCCAGGT ACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAA GCGCAACCCCTGAATCCGGTCCA GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTA CCGAACCGTCCGAAGGTAGCGCA CCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTT CTGAAAGCGCAACCCCGGAATCC GGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTA GCCCTGCTGGCTCTCCAACCTCC ACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAG GTAGCGAACCGGCAACCTCCGGT TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCC CAGGTACCTCTACTGAACCGTCT GAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG CACCAGGTACTTCTACCGAACCG TCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCA GCGCTCCAGGTACCTCTACCGAA CCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGG GTAGCGCACCAGGTAGCCCAGCA GGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCG AGGGTAGCGCACCAGGTACCTCT GAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCT CCGGCTCTGAGACTCCAGGTACC TCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGGT ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTG AACCGTCCGAGGGCAGCGCACCA GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGG CTGGCTCTCCGACTTCCACCGAG GAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCC CGGCAGGCTCTCCGACCTCTACT GAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTA CCTCTACCGAACCGTCTGAGGGC AGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAG GTAGCGAACCTGCTACCTCCGGC TCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTC CAGGTAGCGAACCTGCAACCTCT GGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTG GCCCAGGTACTTCTACTGAACCG TCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCA CCGAAGAAGGTACCTCTGAAAGC GCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT CTGAAACCCCAGGTACTTCTGAA AGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGA CTTCCACCGAGGAAGGTAGCCCG GCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTT CCGAGGGCAGCGCACCAGGTACT TCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCG CTACTCCTGAATCCGGTCCAGGT ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGG CTACTTCTGGTTCTGAAACCCCA GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAG CAGGCTCTCCGACTTCCACTGAG GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT CTACTGAACCTTCTGAGGGCAGC GCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA CCTCTGAAAGCGCTACTCCTGAA TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAG GTTAA FIX- MQRVNMIMAESPGLITI 606ATGCAGCGCGTGAACATGATCAT 607 XTEN_AF864, CLLGYLLSAECTVFLDHGGCAGAATCACCAGGCCTCATCA  pCW0564 ENANKILNRPKRYNSGKCCATCTGCCTTTTAGGATATCTA  LEEFVQGNLERECMEEK CTCAGTGCTGAATGTACAGTTTT CSFEEAREVFENTERTT TCTTGATCATGAAAACGCCAACA  EFWKQYVDGDQCESNPCAAATTCTGAATCGGCCAAAGAGG  LNGGSCKDDINSYECWC TATAATTCAGGTAAATTGGAAGA PFGFEGKNCELDVTCNI GTTTGTTCAAGGGAACCTTGAGA  KNGRCEQFCKNSADNKVGAGAATGTATGGAAGAAAAGTGT  VCSCTEGYRLAENQKSC AGTTTTGAAGAAGCACGAGAAGT EPAVPFPCGRVSVSQTS TTTTGAAAACACTGAAAGAACAA  KLTRAETVFPDVDYVNSCTGAATTTTGGAAGCAGTATGTT  TEAETILDNITQSTQSF GATGGAGATCAGTGTGAGTCCAA NDFTRVVGGEDAKPGQF TCCATGTTTAAATGGCGGCAGTT  PWQVVLNGKVDAFCGGSGCAAGGATGACATTAATTCCTAT  IVNEKWIVTAAHCVETG GAATGTTGGTGTCCCTTTGGATT VKITVVAGEHNIEETEH TGAAGGAAAGAACTGTGAATTAG  TEQKRNVIRIIPHHNYNATGTAACATGTAACATTAAGAAT  AAINKYNHDIALLELDE GGCAGATGCGAGCAGTTTTGTAA PLVLNSYVTPICIADKE AAATAGTGCTGATAACAAGGTGG  YTNIFLKFGSGYVSGWGTTTGCTCCTGTACTGAGGGATAT  RVFHKGRSALVLQYLRV CGACTTGCAGAAAACCAGAAGTC PLVDRATCLRSTKFTIY CTGTGAACCAGCAGTGCCATTTC  NNMFCAGFHEGGRDSCQCATGTGGAAGAGTTTCTGTTTCA  GDSGGPHVTEVEGTSFL CAAACTTCTAAGCTCACCCGTGC TGIISWGEECAMKGKYG TGAGACTGTTTTTCCTGATGTGG  IYTKVSRYVNWIKEKTKACTATGTAAATTCTACTGAAGCT  LTGGSTSESPSGTAPGT GAAACCATTTTGGATAACATCAC SPSGESSTAPGSTSESP TCAAAGCACCCAATCATTTAATG  SGTAPGSTSESPSGTAPACTTCACTCGGGTTGTTGGTGGA  GTSTPESGSASPGTSTP GAAGATGCCAAACCAGGTCAATT ESGSASPGSTSESPSGT CCCTTGGCAGGTTGTTTTGAATG  APGSTSESPSGTAPGTSGTAAAGTTGATGCATTCTGTGGA  PSGESSTAPGSTSESPS GGCTCTATCGTTAATGAAAAATG GTAPGTSPSGESSTAPG GATTGTAACTGCTGCCCACTGTG  TSPSGESSTAPGSTSSTTTGAAACTGGTGTTAAAATTACA  AESPGPGTSPSGESSTA GTTGTCGCAGGTGAACATAATAT PGTSPSGESSTAPGSTS TGAGGAGACAGAACATACAGAGC  STAESPGPGTSTPESGSAAAAGCGAAATGTGATTCGAATT  ASPGTSTPESGSASPGS ATTCCTCACCACAACTACAATGC TSESPSGTAPGSTSESP AGCTATTAATAAGTACAACCATG  SGTAPGTSTPESGSASPACATTGCCCTTCTGGAACTGGAC  GSTSSTAESPGPGTSTP GAACCCTTAGTGCTAAACAGCTA ESGSASPGSTSESPSGT CGTTACACCTATTTGCATTGCTG  APGTSPSGESSTAPGSTACAAGGAATACACGAACATCTTC  SSTAESPGPGTSPSGES CTCAAATTTGGATCTGGCTATGT STAPGTSTPESGSASPG AAGTGGCTGGGGAAGAGTCTTCC  STSSTAESPGPGSTSSTACAAAGGGAGATCAGCTTTAGTT  AESPGPGSTSSTAESPG CTTCAGTACCTTAGAGTTCCACT PGSTSSTAESPGPGTSP TGTTGACCGAGCCACATGTCTTC  SGESSTAPGSTSESPSGGATCTACAAAGTTCACCATCTAT  TAPGSTSESPSGTAPGT AACAACATGTTCTGTGCTGGCTT STPESGPGGGGASASGA CCATGAAGGAGGTAGAGATTCAT  PSTGGGGSESPSGTAPGGTCAAGGAGATAGTGGGGGACCC  STSESPSGTAPGSTSES CATGTTACTGAAGTGGAAGGGAC PSGTAPGSTSESPSGTA CAGTTTCTTAACTGGAATTATTA  PGSTSESPSGTAPGSTSGCTGGGGTGAAGAGTGTGCAATG  ESPSGTAPGTSTPESGS AAAGGCAAATATGGAATATATAC ASPGTSPSGESSTAPGT CAAGGTATCCCGGTATGTCAACT  SPSGESSTAPGSTSSTAGGATTAAGGAAAAAACAAAGCTC  ESPGPGTSPSGESSTAP ACTGGAGGTTCTACCAGCGAATC GTSTPESGSASPGSTSE TCCTTCTGGCACCGCTCCAGGTA  SPSGTAPGSTSESPSGTCCTCTCCTAGCGGCGAATCTTCT  APGTSPSGESSTAPGST ACCGCTCCAGGTTCTACTAGCGA SESPSGTAPGTSTPESG ATCTCCTTCTGGCACTGCACCAG  SASPGTSTPESGSASPGGTTCTACTAGCGAATCCCCGTCT  STSESPSGTAPGTSTPE GGTACTGCTCCAGGTACTTCTAC SGSASPGSTSSTAESPG TCCTGAAAGCGGTTCCGCTTCTC  PGSTSESPSGTAPGSTSCAGGTACCTCTACTCCGGAAAGC  ESPSGTAPGTSPSGESS GGTTCTGCATCTCCAGGTTCTAC TAPGSTSSTAESPGPGT CAGCGAATCTCCTTCTGGCACCG  SPSGESSTAPGTSTPESCTCCAGGTTCTACTAGCGAATCC  GSASPGTSPSGESSTAP CCGTCTGGTACCGCACCAGGTAC GTSPSGESSTAPGTSPS TTCTCCTAGCGGCGAATCTTCTA  GESSTAPGSTSSTAESPCCGCACCAGGTTCTACTAGCGAA  GPGSTSSTAESPGPGTS TCTCCGTCTGGCACTGCTCCAGG PSGESSTAPGSSPSAST TACTTCTCCTAGCGGTGAATCTT  GTGPGSSTPSGATGSPGCTACCGCTCCAGGTACTTCCCCT  SSTPSGATGSPG AGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAG AATCTCCGGGCCCAGGTACCTCT CCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAGCGGTG AATCTTCTACCGCTCCAGGTTCT ACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGG AAAGCGGCTCTGCATCTCCAGGT ACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCG AATCTCCTTCTGGCACTGCACCA GGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTA CCCCTGAAAGCGGTTCCGCTTCT CCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCT CTACTCCGGAAAGCGGCTCTGCA TCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTA CTTCTCCGAGCGGTGAATCTTCT ACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG GTACTTCTCCGAGCGGTGAATCT TCTACTGCTCCAGGTACCTCTACTCCTGAAAGCGGTTCTGCATCTC CAGGTTCCACTAGCTCTACCGCA GAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTG GCCCAGGTTCTACTAGCTCTACT GCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTC CTGGTCCAGGTACCTCCCCGAGC GGTGAATCTTCTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTG GCACTGCACCAGGTTCTACCAGC GAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCG GTCCGGGGGGGGGGGGTGCAAGC GCAAGCGGCGCGCCAAGCACGGGAGGGGGGGGTAGCGAATCTCCTT CTGGTACCGCTCCAGGTTCTACC AGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTC CTTCTGGTACTGCACCAGGTTCT ACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAAT CCCCGTCTGGTACTGCTCCAGGT TCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTACTTCTACTC CGGAAAGCGGTTCCGCATCTCCA GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTC CTAGCGGCGAATCTTCTACTGCT CCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTT CCCCGAGCGGTGAATCTTCTACT GCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTT CTACCAGCGAATCTCCTTCTGGC ACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG GTACTTCTCCTAGCGGCGAATCT TCTACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCAC CAGGTACTTCTACCCCGGAAAGC GGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCAT CTCCAGGTTCTACTAGCGAATCT CCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCG CTTCTCCAGGTTCCACTAGCTCT ACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTG GCACCGCTCCAGGTTCTACTAGC GAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAAT CTTCTACCGCACCAGGTTCTACC AGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTG AATCTTCTACTGCACCAGGTACT TCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTCCCCTAGCG GCGAATCTTCTACTGCTCCAGGT ACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTA GCGGTGAATCTTCTACCGCACCA GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCA GCTCTACTGCTGAATCTCCTGGT CCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTA GCCCTTCTGCTTCCACCGGTACC GGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTA GCTCTACTCCGTCTGGTGCAACC GGCTCCCCAGGTTAA FIX-MQRVNMIMAESPGLITI 608 ATGCAGCGCGTGAACATGATCAT 609 XTEN_AG864,CLLGYLLSAECTVFLDH GGCAGAATCACCAGGCCTCATCA  pCW0565 ENANKILNRPKRYNSGKCCATCTGCCTTTTAGGATATCTA  LEEFVQGNLERECMEEK CTCAGTGCTGAATGTACAGTTTT CSFEEAREVFENTERTT TCTTGATCATGAAAACGCCAACA  EFWKQYVDGDQCESNPCAAATTCTGAATCGGCCAAAGAGG  LNGGSCKDDINSYECWC TATAATTCAGGTAAATTGGAAGA PFGFEGKNCELDVTCNI GTTTGTTCAAGGGAACCTTGAGA  KNGRCEQFCKNSADNKVGAGAATGTATGGAAGAAAAGTGT  VCSCTEGYRLAENQKSC AGTTTTGAAGAAGCACGAGAAGT EPAVPFPCGRVSVSQTS TTTTGAAAACACTGAAAGAACAA  KLTRAETVFPDVDYVNSCTGAATTTTGGAAGCAGTATGTT  TEAETILDNITQSTQSF GATGGAGATCAGTGTGAGTCCAA NDFTRVVGGEDAKPGQF TCCATGTTTAAATGGCGGCAGTT  PWQVVLNGKVDAFCGGSGCAAGGATGACATTAATTCCTAT  IVNEKWIVTAAHCVETG GAATGTTGGTGTCCCTTTGGATT VKITVVAGEHNIEETEH TGAAGGAAAGAACTGTGAATTAG  TEQKRNVIRIIPHHNYNATGTAACATGTAACATTAAGAAT  AAINKYNHDIALLELDE GGCAGATGCGAGCAGTTTTGTAA PLVLNSYVTPICIADKE AAATAGTGCTGATAACAAGGTGG  YTNIFLKFGSGYVSGWGTTTGCTCCTGTACTGAGGGATAT  RVFHKGRSALVLQYLRV CGACTTGCAGAAAACCAGAAGTC PLVDRATCLRSTKFTIY CTGTGAACCAGCAGTGCCATTTC  NNMFCAGFHEGGRDSCQCATGTGGAAGAGTTTCTGTTTCA  GDSGGPHVTEVEGTSFL CAAACTTCTAAGCTCACCCGTGC TGIISWGEECAMKGKYG TGAGACTGTTTTTCCTGATGTGG  IYTKVSRYVNWIKEKTKACTATGTAAATTCTACTGAAGCT  LTGGASPGTSSTGSPGS GAAACCATTTTGGATAACATCAC SPSASTGTGPGSSPSAS TCAAAGCACCCAATCATTTAATG  TGTGPGTPGSGTASSSPACTTCACTCGGGTTGTTGGTGGA  GSSTPSGATGSPGSNPS GAAGATGCCAAACCAGGTCAATT ASTGTGPGASPGTSSTG CCCTTGGCAGGTTGTTTTGAATG  SPGTPGSGTASSSPGSSGTAAAGTTGATGCATTCTGTGGA  TPSGATGSPGTPGSGTA GGCTCTATCGTTAATGAAAAATG SSSPGASPGTSSTGSPG GATTGTAACTGCTGCCCACTGTG  ASPGTSSTGSPGTPGSGTTGAAACTGGTGTTAAAATTACA  TASSSPGSSTPSGATGS GTTGTCGCAGGTGAACATAATAT PGASPGTSSTGSPGTPG TGAGGAGACAGAACATACAGAGC  SGTASSSPGSSTPSGATAAAAGCGAAATGTGATTCGAATT  GSPGSNPSASTGTGPGS ATTCCTCACCACAACTACAATGC SPSASTGTGPGSSTPSG AGCTATTAATAAGTACAACCATG  ATGSPGSSTPSGATGSPACATTGCCCTTCTGGAACTGGAC  GASPGTSSTGSPGASPG GAACCCTTAGTGCTAAACAGCTA TSSTGSPGASPGTSSTG CGTTACACCTATTTGCATTGCTG  SPGTPGSGTASSSPGASACAAGGAATACACGAACATCTTC  PGTSSTGSPGASPGTSS CTCAAATTTGGATCTGGCTATGT TGSPGASPGTSSTGSPG AAGTGGCTGGGGAAGAGTCTTCC  SSPSASTGTGPGTPGSGACAAAGGGAGATCAGCTTTAGTT  TASSSPGASPGTSSTGS CTTCAGTACCTTAGAGTTCCACT PGASPGTSSTGSPGASP TGTTGACCGAGCCACATGTCTTC  GTSSTGSPGSSTPSGATGATCTACAAAGTTCACCATCTAT  GSPGSSTPSGATGSPGA AACAACATGTTCTGTGCTGGCTT SPGTSSTGSPGTPGSGT CCATGAAGGAGGTAGAGATTCAT  ASSSPGSSTPSGATGSPGTCAAGGAGATAGTGGGGGACCC  GSSTPSGATGSPGSSTP CATGTTACTGAAGTGGAAGGGAC SGATGSPGSSPSASTGT CAGTTTCTTAACTGGAATTATTA  GPGASPGTSSTGSPGASGCTGGGGTGAAGAGTGTGCAATG  PGTSSTGSPGTPGSGTA AAAGGCAAATATGGAATATATAC SSSPGASPGTSSTGSPG CAAGGTATCCCGGTATGTCAACT  ASPGTSSTGSPGASPGTGGATTAAGGAAAAAACAAAGCTC  SSTGSPGASPGTSSTGS ACTGGAGGTGCTTCCCCGGGCAC PGTPGSGTASSSPGSST CAGCTCTACTGGTTCTCCAGGTT  PSGATGSPGTPGSGTASCTAGCCCGTCTGCTTCTACTGGT  SSPGSSTPSGATGSPGT ACTGGTCCAGGTTCTAGCCCTTC PGSGTASSSPGSSTPSG TGCTTCCACTGGTACTGGTCCAG  ATGSPGSSTPSGATGSPGTACCCCGGGTAGCGGTACCGCT  GSSPSASTGTGPGSSPS TCTTCTTCTCCAGGTAGCTCTAC ASTGTGPGASPGTSSTG TCCGTCTGGTGCTACCGGCTCTC  SPGTPGSGTASSSPGSSCAGGTTCTAACCCTTCTGCATCC  TPSGATGSPGSSPSAST ACCGGTACCGGCCCAGGTGCTTC GTGPGSSPSASTGTGPG TCCGGGCACCAGCTCTACTGGTT  ASPGTSSTGSPGASPGTCTCCAGGTACCCCGGGCAGCGGT  SSTGSPGSSTPSGATGS ACCGCATCTTCTTCTCCAGGTAG PGSSPSASTGTGPGASP CTCTACTCCTTCTGGTGCAACTG  GTSSTGSPGSSPSASTGGTTCTCCAGGTACTCCTGGCAGC  TGPGTPGSGTASSSPGS GGTACCGCTTCTTCTTCTCCAGG STPSGATGSPGSSTPSG TGCTTCTCCTGGTACTAGCTCTA  ATGSPGASPGTSSTGSPCTGGTTCTCCAGGTGCTTCTCCG  G GGCACTAGCTCTACTGGTTCTCC AGGTACCCCGGGTAGCGGTACTG CTTCTTCCTCTCCAGGTAGCTCT ACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCA GCTCTACCGGTTCTCCAGGTACC CCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGT CTGGTGCTACCGGCTCTCCAGGT TCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCTT CTGCTTCCACCGGTACTGGCCCA GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTA CTCCTTCTGGTGCAACTGGCTCT CCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCAT CCCCTGGCACTAGCTCTACTGGT TCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTA CTCCTGGCAGCGGTACCGCTTCT TCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAG GTGCTTCTCCGGGCACTAGCTCT ACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTC CAGGTTCTAGCCCTTCTGCATCT ACTGGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCT CTCCAGGTGCATCTCCGGGCACT AGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTG GTTCTCCAGGTGCTTCTCCTGGT ACCAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAA CCGGTTCCCCAGGTAGCTCTACT CCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCT CTACCGGTTCTCCAGGTACCCCG GGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTG GTGCTACCGGTTCCCCAGGTAGC TCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACTCCGT CTGGTGCAACCGGCTCCCCAGGT TCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGG GCACCAGCTCTACTGGTTCTCCA GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCTG GCAGCGGTACTGCATCTTCCTCT CCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCAT CTCCGGGCACTAGCTCTACTGGT TCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTG CTTCTCCTGGTACCAGCTCTACT GGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAG GTAGCTCTACTCCGTCTGGTGCT ACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTC CAGGTAGCTCTACCCCGTCTGGT GCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACCCCTTCT GGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTG GCTCCCCAGGTTCTAGCCCTTCT GCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCATCTACTG GTACTGGTCCAGGTGCATCCCCG GGCACTAGCTCTACCGGTTCTCCAGGTACTCCTGGTAGCGGTACTG CTTCTTCTTCTCCAGGTAGCTCT ACTCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCAT CCACCGGTACCGGCCCAGGTTCT AGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTA CTAGCTCTACTGGTTCTCCAGGT GCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTC CGTCTGGTGCAACCGGCTCTCCA GGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCC CTGGTACCAGCTCTACCGGTTCT CCAGGTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCC CTGGCAGCGGTACCGCATCTTCC TCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTA GCTCTACTCCTTCTGGTGCTACT GGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAG GTTAA PC5, pBC0037, MGWGSRCCCPGRLDLLC 610atgggctgggggagccgctgctg 611 pBC0038 VLALLGGCLLPVCRTRVctgcccgggacgtttggacctgc YTNHWAVKIAGGFPEAN tgtgcgtgctggcgctgctcggg RIASKYGFINIGQIGAL ggctgcctgctccccgtgtgtcg  KDYYHFYHSRTIKRSVIgacgcgcgtctacaccaaccact  SSRGTHSFISMEPKVEW gggcagtcaaaatcgccgggggc IQQQVVKKRTKRDYDFS ttcccggaggccaaccgtatcgc  RAQSTYFNDPKWPSMWYcagcaagtacggattcatcaaca  MHCSDNTHPCQSDMNIE taggacagataggggccctgaag GAWKRGYTGKNIVVTIL gactactaccacttctaccatag  DDGIERTHPDLMQNYDAcaggacgattaaaaggtcagtta  LASCDVNGNDLDPMPRY tctcgagcagagggacccacagt DASNENKHGTRCAGEVA ttcatttcaatggaaccaaaggt  AAANNSHCTVGIAFNAKggaatggatccaacagcaagtgg  IGGVRMLDGDVTDMVEA taaaaaagcggacaaagagggat KSVSFNPQHVHIYSASW tatgacttcagtcgtgcccagtc  GPDDDGKTVDGPAPLTRtacctatttcaatgatcccaagt  QAFENGVRMGRRGLGSV ggcccagcatgtggtatatgcac FVWASGNGGRSKDHCSC tgcagtgacaatacacatccctg  DGYTNSIYTISISSTAEccagtctgacatgaatatcgaag  SGKKPWYLEECSSTLAT gagcctggaagagaggctacacg TYSSGESYDKKIITTDL ggaaagaacattgtggtcactat  RQRCTDNHTGTSASAPMcctggatgacggaattgagagaa  AAGIIALALEANPFLTW cccatccagatctgatgcaaaac RDVQHVIVRTSRAGHLN tacgatgctctggcaagttgcga  ANDWKTNAAGFKVSHLYcgtgaatgggaatgacttggacc  GFGLMDAEAMVMEAEKW caatgcctcgttatgatgcaagc TTVPRQHVCVESTDRQI aacgagaacaagcatgggactcg  KTIRPNSAVRSIYKASGctgtgctggagaagtggcagccg  CSDNPNRHVNYLEHVVV ctgcaaacaattcgcactgcaca RITITHPRRGDLAIYLT gtcggaattgctttcaacgccaa  SPSGTRSQLLANRLFDHgatcggaggagtgcgaatgctgg  SMEGFKNWEFMTIHCWG acggagatgtcacggacatggtt ERAAGDWVLEVYDTPSQ gaagcaaaatcagttagcttcaa  LRNFKTPGKLKEWSLVLcccccagcacgtgcacatttaca  YGTSVQPYSPTNEFPKV gcgccagctggggcccggatgat ERFRYSRVEDPTDDYGT gatggcaagactgtggacggacc  EDYAGPCDPECSEVGCDagcccccctcacccggcaagcct  GPGPDHCNDCLHYYYKL ttgaaaacggcgttagaatgggg KNNTRICVSSCPPGHYH cggagaggcctcggctctgtgtt  ADKKRCRKCAPNCESCFtgtttgggcatctggaaatggtg  GSHGDQCMSCKYGYFLN gaaggagcaaagaccactgctcc EETNSCVTHCPDGSYQD tgtgatggctacaccaacagcat  TKKNLCRKCSENCKTCTctacaccatctccatcagcagca  EFHNCTECRDGLSLQGS ctgcagaaagcggaaagaaacct RCSVSCEDGRYFNGQDC tggtacctggaagagtgttcatc  QPCHRFCATCAGAGADGcacgctggccacaacctacagca  CINCTEGYFMEDGRCVQ gcggggagtcctacgataagaaa SCSISYYFDHSSENGYK atcatcactacagatctgaggca  SCKKCDISCLTCNGPGFgcgttgcacggacaaccacactg  KNCTSCPSGYLLDLGMC ggacgtcagcctcagcccccatg QMGAICKDAIEESWAEG gctgcaggcatcattgcgctggc  GFCMLVKKNNLCQRKVLcctggaagccaatccgtttctga  QQLCCKTCTFQG cctggagagacgtacagcatgtt attgtcaggacttcccgtgcggg  acatttgaacgctaatgactgga aaaccaatgctgctggttttaag  gtgagccatctttatggatttgg actgatggacgcagaagccatggtgatggaggcagagaagtggacc accgttccccggcagcacgtgtg tgtggagagcacagaccgacaaatcaagacaatccgccctaacagt gcagtgcgctccatctacaaagc ttcaggctgctcggataaccccaaccgccatgtcaactacctggag cacgtcgttgtgcgcatcaccat cacccaccccaggagaggagacctggccatctacctgacctcgccc tctggaactaggtctcagctttt ggccaacaggctatttgatcactccatggaaggattcaaaaactgg gagttcatgaccattcattgctg gggagaaagagctgctggtgactgggtccttgaagtttatgatact ccctctcagctaaggaactttaa gactccaggtaaattgaaagaatggtctttggtcctctacggcACC TCCGTGCAGCCATATTCaccaac caatgaatttccgaaagtggaacggttccgctatagccgagttgaa gaccccacagacgactatggcac agaggattatgcaggtccctgcgaccctgagtgcagtgaggttggc tgtgacgggccaggaccagacca ctgcaatgactgtttgcactactactacaagctgaaaaacaatacc aggatctgtgtctccagctgccc ccctggccactaccacgccgacaagaagcgctgcaggaagtgtgcc cccaactgtgagtcctgctttgg gagccatggtgaccaatgcatgtcctgcaaatatggatactttctg aatgaagaaaccaacagctgtgt tactcactgccctgatgggtcatatcaggataccaagaaaaatctt tgccggaaatgcagtgaaaactg caagacatgtactgaattccataactgtacagaatgtagggatggg ttaagcctgcagggatcccggtg ctctgtctcctgtgaagatggacggtatttcaacggccaggactgc cagccctgccaccgcttctgcgc cacttgtgctggggcaggagctgatgggtgcattaactgcacagag ggctacttcatggaggatgggag atgcgtgcagagctgtagtatcagctattactttgaccactcttca gagaatggatacaaatcctgcaa aaaatgtgatatcagttgtttgacgtgcaatggcccaggattcaag aactgtacaagctgccctagtgg gtatctcttagacttaggaatgtgtcaaatgggagccatttgcaag gatgcaacggaagagtcctgggc ggaaggaggcttctgtatgcttgtgaaaaagaacaatctgtgccaa cggaaggttcttcaacaactttg ctgcaaaacatgtacatttcaaggctga

Example 24: Construction of FIX-/FXI/-XTEN

Construction of FIX-TEV-XTEN_AE864 Genes and Vectors

The cloning vector containing the gene encoding FIX was purchased fromOriGene (SC126517). PCR reactions were performed to abolish two BbsIrestriction sites within the FIX coding region. The resulting FIX codingregion was then amplified using primers that introduced NheI andSfiI-TEV-BsaI sequences on the 5′ and 3′ end respectively. The digestedFIX fragment was fused to BsaI/HindIII digested XTEN_AE864 fragment andinserted into NheI/HindIII digested pSecTag2C expression vector. Theligated DNA mixture was electroporated into XL1-Blue bacterial cells.Transformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The final construct is pCW0648.001which encodes the FIX-TEV-XTEN_AE864 protein (Table 25).

Construction of FIX-/FXI/-XTEN_AE864 Genes and Vectors

The TEV site was removed by digesting the pCW0648 expression vector withSfiI and BsaI. Oligos containing sequences that encode SfiI-KLTRAET-BsaI(‘KLTRAET’ disclosed as SEQ ID NO: 6), SfiI-DFTRVVG-BsaI (‘DFTRVVG’disclosed as SEQ ID NO: 88), or SfiI-/FXI/-BsaI were annealed andligated with the digested pCW0648 vector. The ligated DNA mixture waselectroporated into XL1-Blue bacterial cells. Transformants werescreened by DNA miniprep and the desired constructs were confirmed byDNA sequencing. The resulting expression vectors encodeFIX-KLTRAET-XTEN_AE864 (pCW0735) (‘KLTRAET’ disclosed as SEQ ID NO: 6),FIX-DFTRVVG-XTEN_AE864 (pCW0736) (‘DFTRVVG’ disclosed as SEQ ID NO: 88)and FIX-/FXI/-XTEN_AE864 (pCW0737).

Construction of Expression Vectors Encoding FIX-/FXI/-XTEN_AE864 GenesUsing Millipore Plasmids

Expression vector pCW0735 was digested with NheI and SalI. The resulting4181 bp fragment included nucleotides that encode theFIX-KLTRAET-XTEN_AE864 protein (‘KLTRAET’ disclosed as SEQ ID NO: 6).This fragment was ligated with NheI/SalI digested CET1019-HD-puro(Millipore) or DC HD-puro (Millipore). Both CET1019-HD-puro andDC-HD-puro feature dual cassettes where a CMV promoter lies upstream ofthe gene insertion site, CET1019-HD-puro also contains a UCOE elementupstream of the promoter. The ligated DNA mixture was electroporatedinto XL1-Blue bacterial cells. Transformants were screened by DNAminiprep and the desired constructs were confirmed by DNA sequencing.The resulting expression vectors were pBC0033(DC-HD-puro-FIX-KLTRAET-XTEN_AE864 (‘KLTRAET’ disclosed as SEQ ID NO:6)) and pBC0034 (CET1019-HD-puro-FIX-KLTRAET-XTEN_AE864 (‘KLTRAET’disclosed as SEQ ID NO: 6)) (Table 25).

TABLE 25FIX-XTEN with cleavage sequence: amino acid and nucleic acid sequencesSEQ SEQ Amino Acid ID ID Name Sequence NO: Nucleic Acid Sequences NO:FIX-TEV- MQRVNMIMAESPGLITI 612 atgcagcgcgtgaacatgatcatggcagaatcacc 613XTEN_AE864 CLLGYLLSAECTVFLDH aggcctcatcaccatctgccttttaggatatctacENANKILNRPKRYNSGK tcagtgctgaatgtacagtttttcttgatcatgaa  LEEFVQGNLERECMEEKaacgccaacaaaattctgaatcggccaaagaggta  CSFEEAREVFENTERTTtaattcaggtaaattggaagagtttgttcaaggga  EFWKQYVDGDQCESNPCaccttgagagagaatgtatggaagaaaagtgtagt  LNGGSCKDDINSYECWCtttgaagaagcacgagaagtttttgaaaacactga  PFGFEGKNCELDVTCNIaagaacaactgaattttggaagcagtatgttgatg  KNGRCEQFCKNSADNKVgagatcagtgtgagtccaatccatgtttaaatggc  VCSCTEGYRLAENQKSCggcagttgcaaggatgacattaattcctatgaatg  EPAVPFPCGRVSVSQTSttggtgtccctttggatttgaaggaaagaactgtg  KLTRAETVFPDVDYVNSaattagatgtaacatgtaacattaagaatggcaga  TEAETILDNITQSTQSFtgcgagcagttttgtaaaaatagtgctgataacaa  NDFTRVVGGEDAKPGQFggtggtttgctcctgtactgagggatatcgacttg  PWQVVLNGKVDAFCGGScagaaaaccagaagtcctgtgaaccagcagtgcca  IVNEKWIVTAAHCVETGtttccatgtggaagagtttctgtttcacaaacttc  VKITVVAGEHNIEETEHtaagctcacccgtgctgagactgtttttcctgatg  TEQKRNVIRIIPHHNYNtggactatgtaaattctactgaagctgaaaccatt  AAINKYNHDIALLELDEttggataacatcactcaaagcacccaatcatttaa  PLVLNSYVTPICIADKEtgacttcactcgggttgttggtggagaagatgcca  YTNIFLKFGSGYVSGWGaaccaggtcaattcccttggcaggttgttttgaat  RVFHKGRSALVLQYLRVggtaaagttgatgcattctgtggaggctctatcgt  PLVDRATCLRSTKFTIYtaatgaaaaatggattgtaactgctgcccactgtg  NNMFCAGFHEGGRDSCQttgaaactggtgttaaaattacagttgtcgcaggt  GDSGGPHVTEVEGTSFLgaacataatattgaggagacagaacatacagagca  TGIISWGEECAMKGKYGaaagcgaaatgtgattcgaattattcctcaccaca  IYTKVSRYVNWTKEKTKactacaatgcagctattaataagtacaaccatgac  LTGPEGPSENLYFQGGSattgcccttctggaactggacgaacccttagtgct  PGSPAGSPTSTEEGTSEaaacagctacgttacacctatttgcattgctgaca  SATPESGPGTSTEPSEGaggaatacacgaacatcttcctcaaatttggatct  SAPGSPAGSPTSTEEGTggctatgtaagtggctggggaagagtGttccacaa  STEPSEGSAPGTSTEPSagggagatcagctttagttcttcagtaccttagag  EGSAPGTSESATPESGPttccacttgttgaccgagccacatgtctAcgatct  GSEPATSGSETPGSEPAacaaagttcaccatctataacaacatgttctgtgc  TSGSETPGSPAGSPTSTtggcttccatgaaggaggtagagattcatgtcaag  EEGTSESATPESGPGTSgagatagtgggggaccccatgttactgaagtggaa  TEPSEGSAPGTSTEPSEgggaccagtttcttaactggaattattagctgggg  GSAPGSPAGSPTSTEEGtgaagagtgtgcaatgaaaggcaaatatggaatat  TSTEPSEGSAPGTSTEPataccaaggtatcccggtatgtcaactggattaag  SEGSAPGTSESATPESGgaaaaaacaaagctcactGGCCCAGAAGGCCCAtc  PGTSTEPSEGSAPGTSEcgaaaatctgtattttcagggtGGGTCTCCAGGTT  SATPESGPGSEPATSGSCTCCAGCCGGGTCCCCAACTTCGACCGAGGAAGGG  ETPGTSTEPSEGSAPGTACCTCCGAGTCAGCTACCCCGGAGTCCGGTCCTGG  STEPSEGSAPGTSESATCACCTCCACCGAACCATCGGAGGGCAGCGCCCCTG  PESGPGTSESATPESGPGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAGAG  GSPAGSPTSTEEGTSESGGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCC  ATPESGPGSEPATSGSETGGTACTAGTACTGAACCATCCGAGGGGTCAGCTC  TPGTSESATPESGPGTSCAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGA  TEPSEGSAPGTSTEPSECCGGGCTCAGAGCCCGCCACGAGTGGCAGTGAAAC  GSAPGTSTEPSEGSAPGTCCAGGCTCAGAACCCGCCACTAGTGGGTCAGAGA  TSTEPSEGSAPGTSTEPCTCCAGGCAGCCCTGCCGGATCCCCTACGTCCACC  SEGSAPGTSTEPSEGSAGAGGAGGGAACATCTGAGTCCGCAACACCCGAATC  PGSPAGSPTSTEEGTSTCGGTCCAGGCACCTCCACGGAACCTAGTGAAGGCT  EPSEGSAPGTSESATPECGGCACCAGGTACAAGCACCGAACCTAGCGAGGGC  SGPGSEPATSGSETPGTAGCGCTCCCGGCAGCCCTGCCGGCAGCCCAACCTC  SESATPESGPGSEPATSAACTGAGGAGGGCACCAGTACTGAGCCCAGCGAGG  GSETPGTSESATPESGPGATCAGCACCTGGCACCAGCACCGAACCTAGCGAG  GTSTEPSEGSAPGTSESGGGAGCGCCCCTGGGACTAGCGAGTCAGCTACACC  ATPESGPGSPAGSPTSTAGAGAGCGGGCCTGGAACTTCTACCGAACCCAGTG  EEGSPAGSPTSTEEGSPAGGGATCCGCTCCAGGCACCTCCGAATCCGCAACC  AGSPTSTEEGTSESATPCCCGAATCCGGACCTGGCTCAGAGCCCGCCACCAG  ESGPGTSTEPSEGSAPGCGGGAGCGAAACCCCTGGCACATCCACCGAGCCTA  TSESATPESGPGSEPATGCGAAGGGTCCGCACCCGGCACCAGTACAGAGCCT  SGSETPGTSESATPESGAGCGAGGGATCAGCACCTGGCACCAGTGAATCTGC  PGSEPATSGSETPGTSETACACCAGAGAGCGGCCCTGGAACCTCCGAGTCCG  SATPESGPGTSTEPSEGCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGC  SAPGSPAGSPTSTEEGTTCCCCCACCTCAACAGAAGAGGGGACAAGCGAAAG  SESATPESGPGSEPATSCGCTACGCCTGAGAGTGGCCCTGGCTCTGAGCCAG  GSETPGTSESATPESGPCCACCTCCGGCTCTGAAACCCCTGGCACTAGTGAG  GSPAGSPTSTEEGSPAGTCTGCCACGCCTGAGTCCGGACCCGGGACCTCTAC  SPTSTEEGTSTEPSEGSTGAGCCCTCGGAGGGGAGCGCTCCTGGCACGAGTA  APGTSESATPESGPGTSCAGAACCTTCCGAAGGAAGTGCACCGGGCACAAGC  ESATPESGPGTSESATPACCGAGCCTTCCGAAGGCTCTGCTCCCGGAACCTC  ESGPGSEPATSGSETPGTACCGAACCCTCTGAAGGGTCTGCACCCGGCACGA  SEPATSGSETPGSPAGSGCACCGAACCCAGCGAAGGGTCAGCGCCTGGGACC  PTSTEEGTSTEPSEGSATCAACAGAGCCCTCGGAAGGATCAGCGCCTGGAAG  PGTSTEPSEGSAPGSEPCCCTGCAGGGAGTCCAACTTCCACGGAAGAAGGAA  ATSGSETPGTSESATPECGTCTACAGAGCCATCAGAGGGGTCCGCACCAGGT  SGPGTSTEPSEGSAPGSACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGG  SSGTCCGAACCTGCCACCTCCGGCTCTGAAACTCCAG GGACCTCCGAATCTGCCACACCCGAGAGCGGCCCTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAGCACCGAGCCATCCGAGGGATCCGCC CCAGGCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCGCTGGCTCCCCCACCAGTACCG AGGAGGGGTCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCTGCCGGCTCCCCCACAAGTAC CGAAGAGGGCACAAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAGCACAGAGCCTTCCGAGGGG TCCGCACCAGGTACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTCCGGGT CTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCCACTTCCGG CTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACAGAGCCCTCT GAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCA CACCCGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCGC CACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGAGGAGGGATCTCCTGCTGGC TCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGCCATCCGAGGGATCAGCTCCCGGCACCTCAGAGT CGGCAACCCCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGA ATCAGCAACACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGATCAGAA CCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCACTGAGGAGGGCACCA GCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGTTCAGCACCCGGCTC TGAGCCGGCCACAAGTGGCAGTGAGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGC ACTAGTACCGAGCCCTCTGAAGGCAGTGCGCCAGGTTCGTCTTCATAA FIX- MQRVNMIMAESPGLITI 614atgcagcgcgtgaacatgatcatggcagaatcacc 615 KLTRAET- CLLGYLLSAECTVFLDHaggcctcatcaccatctgccttttaggatatctac  XTEN_AE864 ENANKILNRPKRYNSGKtcagtgctgaatgtacagtttttcttgatcatgaa  (‘KLTRAET’ LEEFVQGNLERECMEEKaacgccaacaaaattctgaatcggccaaagaggta  disclosed as CSFEEAREVFENTERTTtaattcaggtaaattggaagagtttgttcaaggga  SEQ ID NO: 6) EFWKQYVDGDQCESNPCaccttgagagagaatgtatggaagaaaagtgtagt  LNGGSCKDDINSYECWCtttgaagaagcacgagaagtttttgaaaacactga  PFGFEGKNCELDVTCNIaagaacaactgaattttggaagcagtatgttgatg  KNGRCEQFCKNSADNKVgagatcagtgtgagtccaatccatgtttaaatggc  VCSCTEGYRLAENQKSCggcagttgcaaggatgacattaattcctatgaatg  EPAVPFPCGRVSVSQTSttggtgtccctttggatttgaaggaaagaactgtg  KLTRAETVFPDVDYVNSaattagatgtaacatgtaacattaagaatggcaga  TEAETILDNITQSTQSFtgcgagcagttttgtaaaaatagtgctgataacaa  NDFTRVVGGEDAKPGQFggtggtttgctcctgtactgagggatatcgacttg  PWQVVLNGKVDAFCGGScagaaaaccagaagtcctgtgaaccagcagtgcca  IVNEKWIVTAAHCVETGtttccatgtggaagagtttctgtttcacaaacttc  VKITVVAGEHNIEETEHtaagctcacccgtgctgagactgtttttcctgatg  TEQKRNVIRIIPHHNYNtggactatgtaaattctactgaagctgaaaccatt  AAINKYNHDIALLELDEttggataacatcactcaaagcacccaatcatttaa  PLVLNSYVTPICIADKEtgacttcactcgggttgttggtggagaagatgcca  YTNIFLKFGSGYVSGWGaaccaggtcaattcccttggcaggttgttttgaat  RVFHKGRSALVLQYLRVggtaaagttgatgcattctgtggaggctctatcgt  PLVDRATCLRSTKFTIYtaatgaaaaatggattgtaactgctgcccactgtg  NNMFCAGFHEGGRDSCQttgaaactggtgttaaaattacagttgtcgcaggt  GDSGGPHVTEVEGTSFLgaacataatattgaggagacagaacatacagagca  TGIISWGEECAMKGKYGaaagcgaaatgtgattcgaattattcctcaccaca  IYTKVSRYVNWIKEKTKactacaatgcagctattaataagtacaaccatgac  LTGPEGPSKLTRAETGSattgcccttctggaactggacgaacccttagtgct  PGSPAGSPTSTEEGTSEaaacagctacgttacacctatttgcattgctgaca  SATPESGPGTSTEPSEGaggaatacacgaacatcttcctcaaatttggatct  SAPGSPAGSPTSTEEGTggctatgtaagtggctggggaagagtGttccacaa  STEPSEGSAPGTSTEPSagggagatcagctttagttcttcagtaccttagag  EGSAPGTSESATPESGPttccacttgttgaccgagccacatgtctAcgatct  GSEPATSGSETPGSEPAacaaagttcaccatctataacaacatgttctgtgc  TSGSETPGSPAGSPTSTtggcttccatgaaggaggtagagattcatgtcaag  EEGTSESATPESGPGTSgagatagtgggggaccccatgttactgaagtggaa  TEPSEGSAPGTSTEPSEgggaccagtttcttaactggaattattagctgggg  GSAPGSPAGSPTSTEEGtgaagagtgtgcaatgaaaggcaaatatggaatat  TSTEPSEGSAPGTSTEPataccaaggtatcccggtatgtcaactggattaag  SEGSAPGTSESATPESGgaaaaaacaaagctcactGGCCCAGAAGGCCCAtc  PGTSTEPSEGSAPGTSEcaagctAacGcgtgcGgagacAGGGTCTCCAGGTT  SATPESGPGSEPATSGSCTCCAGCCGGGTCCCCAACTTCGACCGAGGAAGGG  ETPGTSTEPSEGSAPGTACCTCCGAGTCAGCTACCCCGGAGTCCGGTCCTGG  STEPSEGSAPGTSESATCACCTCCACCGAACCATCGGAGGGCAGCGCCCCTG  PESGPGTSESATPESGPGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAGAG  GSPAGSPTSTEEGTSESGGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCC  ATPESGPGSEPATSGSETGGTACTAGTACTGAACCATCCGAGGGGTCAGCTC  TPGTSESATPESGPGTSCAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGA  TEPSEGSAPGTSTEPSECCGGGCTCAGAGCCCGCCACGAGTGGCAGTGAAAC  GSAPGTSTEPSEGSAPGTCCAGGCTCAGAACCCGCCACTAGTGGGTCAGAGA  TSTEPSEGSAPGTSTEPCTCCAGGCAGCCCTGCCGGATCCCCTACGTCCACC  SEGSAPGTSTEPSEGSAGAGGAGGGAACATCTGAGTCCGCAACACCCGAATC  PGSPAGSPTSTEEGTSTCGGTCCAGGCACCTCCACGGAACCTAGTGAAGGCT  EPSEGSAPGTSESATPECGGCACCAGGTACAAGCACCGAACCTAGCGAGGGC  SGPGSEPATSGSETPGTAGCGCTCCCGGCAGCCCTGCCGGCAGCCCAACCTC  SESATPESGPGSEPATSAACTGAGGAGGGCACCAGTACTGAGCCCAGCGAGG  GSETPGTSESATPESGPGATCAGCACCTGGCACCAGCACCGAACCTAGCGAG  GTSTEPSEGSAPGTSESGGGAGCGCCCCTGGGACTAGCGAGTCAGCTACACC  ATPESGPGSPAGSPTSTAGAGAGCGGGCCTGGAACTTCTACCGAACCCAGTG  EEGSPAGSPTSTEEGSPAGGGATCCGCTCCAGGCACCTCCGAATCCGCAACC  AGSPTSTEEGTSESATPCCCGAATCCGGACCTGGCTCAGAGCCCGCCACCAG  ESGPGTSTEPSEGSAPGCGGGAGCGAAACCCCTGGCACATCCACCGAGCCTA  TSESATPESGPGSEPATGCGAAGGGTCCGCACCCGGCACCAGTACAGAGCCT  SGSETPGTSESATPESGAGCGAGGGATCAGCACCTGGCACCAGTGAATCTGC  PGSEPATSGSETPGTSETACACCAGAGAGCGGCCCTGGAACCTCCGAGTCCG  SATPESGPGTSTEPSEGCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGC  SAPGSPAGSPTSTEEGTTCCCCCACCTCAACAGAAGAGGGGACAAGCGAAAG  SESATPESGPGSEPATSCGCTACGCCTGAGAGTGGCCCTGGCTCTGAGCCAG  GSETPGTSESATPESGPCCACCTCCGGCTCTGAAACCCCTGGCACTAGTGAG  GSPAGSPTSTEEGSPAGTCTGCCACGCCTGAGTCCGGACCCGGGACCTCTAC  SPTSTEEGTSTEPSEGSTGAGCCCTCGGAGGGGAGCGCTCCTGGCACGAGTA  APGTSESATPESGPGTSCAGAACCTTCCGAAGGAAGTGCACCGGGCACAAGC  ESATPESGPGTSESATPACCGAGCCTTCCGAAGGCTCTGCTCCCGGAACCTC  ESGPGSEPATSGSETPGTACCGAACCCTCTGAAGGGTCTGCACCCGGCACGA  SEPATSGSETPGSPAGSGCACCGAACCCAGCGAAGGGTCAGCGCCTGGGACC  PTSTEEGTSTEPSEGSATCAACAGAGCCCTCGGAAGGATCAGCGCCTGGAAG  PGTSTEPSEGSAPGSEPCCCTGCAGGGAGTCCAACTTCCACGGAAGAAGGAA  ATSGSETPGTSESATPECGTCTACAGAGCCATCAGAGGGGTCCGCACCAGGT  SGPGTSTEPSEGSAPGSACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGG  SSGTCCGAACCTGCCACCTCCGGCTCTGAAACTCCAG GGACCTCCGAATCTGCCACACCCGAGAGCGGCCCTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAGCACCGAGCCATCCGAGGGATCCGCC CCAGGCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCGCTGGCTCCCCCACCAGTACCG AGGAGGGGTCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCTGCCGGCTCCCCCACAAGTAC CGAAGAGGGCACAAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAGCACAGAGCCTTCCGAGGGG TCCGCACCAGGTACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTCCGGGT CTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCCACTTCCGG CTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACAGAGCCCTCT GAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCA CACCCGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCGC CACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGAGGAGGGATCTCCTGCTGGC TCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGCCATCCGAGGGATCAGCTCCCGGCACCTCAGAGT CGGCAACCCCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGA ATCAGCAACACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGATCAGAA CCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCACTGAGGAGGGCACCA GCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGTTCAGCACCCGGCTC TGAGCCGGCCACAAGTGGCAGTGAGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGC ACTAGTACCGAGCCCTCTGAAGGCAGTGCGCCAGGTTCGTCTTCATAA FIX- MQRVNMIMAESPGLITI 616atgcagcgcgtgaacatgatcatggcagaatcacc 617 DFTRVVG- CLLGYLLSAECTVFLDHaggcctcatcaccatctgccttttaggatatctac XTEN_AE864 ENANKILNRPKRYNSGKtcagtgctgaatgtacagtttttcttgatcatgaa LEEFVQGNLERECMEEKaacgccaacaaaattctgaatcggccaaagaggta CSFEEAREVFENTERTTtaattcaggtaaattggaagagtttgttcaaggga EFWKQYVDGDQCESNPCaccttgagagagaatgtatggaagaaaagtgtagt LNGGSCKDDINSYECWCtttgaagaagcacgagaagtttttgaaaacactga PFGFEGKNCELDVTCNIaagaacaactgaattttggaagcagtatgttgatg KNGRCEQFCKNSADNKVgagatcagtgtgagtccaatccatgtttaaatggc VCSCTEGYRLAENQKSCggcagttgcaaggatgacattaattcctatgaatg EPAVPFPCGRVSVSQTSttggtgtccctttggatttgaaggaaagaactgtg KLTRAETVFPDVDYVNSaattagatgtaacatgtaacattaagaatggcaga TEAETILDNITQSTQSFtgcgagcagttttgtaaaaatagtgctgataacaa NDFTRVVGGEDAKPGQFggtggtttgctcctgtactgagggatatcgacttg PWQVVLNGKVDAFCGGScagaaaaccagaagtcctgtgaaccagcagtgcca IVNEKWIVTAAHCVETGtttccatgtggaagagtttctgtttcacaaacttc VKITVVAGEHNIEETEHtaagctcacccgtgctgagactgtttttcctgatg TEQKRNVIRIIPHHNYNtggactatgtaaattctactgaagctgaaaccatt AAINKYNHDIALLELDEttggataacatcactcaaagcacccaatcatttaa PLVLNSYVTPICIADKEtgacttcactcgggttgttggtggagaagatgcca YTNIFLKFGSGYVSGWGaaccaggtcaattcccttggcaggttgttttgaat RVFHKGRSALVLQYLRVggtaaagttgatgcattctgtggaggctctatcgt PLVDRATCLRSTKFTIYtaatgaaaaatggattgtaactgctgcccactgtg NNMFCAGFHEGGRDSCQttgaaactggtgttaaaattacagttgtcgcaggt GDSGGPHVTEVEGTSFLgaacataatattgaggagacagaacatacagagca TGIISWGEECAMKGKYGaaagcgaaatgtgattcgaattattcctcaccaca IYTKVSRYVNWIKEKTKactacaatgcagctattaataagtacaaccatgac LTGPEGPSDFTRVVGGSattgcccttctggaactggacgaacccttagtgct PGSPAGSPTSFEEGTSEaaacagctacgttacacctatttgcattgctgaca SATPESGPGTSTEPSEGaggaatacacgaacatcttcctcaaatttggatct SAPGSPAGSPTSTEEGTggctatgtaagtggctggggaagagtGttccacaa STEPSEGSAPGTSTEPSagggagatcagctttagttcttcagtaccttagag EGSAPGTSESATPESGPttccacttgttgaccgagccacatgtctAcgatct GSEPATSGSETPGSEPAacaaagttcaccatctataacaacatgttctgtgc TSGSETPGSPAGSPTSTtggcttccatgaaggaggtagagattcatgtcaag EEGTSESATPESGPGTSgagatagtgggggaccccatgttactgaagtggaa TEPSEGSAPGTSTEPSEgggaccagtttcttaactggaattattagctgggg GSAPGSPAGSPTSTEEGtgaagagtgtgcaatgaaaggcaaatatggaatat TSTEPSEGSAPGTSTEPataccaaggtatcccggtatgtcaactggattaag SEGSAPGTSESATPESGgaaaaaacaaagctcactGGCCCAGAAGGCCCAtc PGTSTEPSEGSAPGTSEcgacttcacAcgggtAgttggCGGGTCTCCAGGTT SATPESGPGSEPATSGSCTCCAGCCGGGTCCCCAACTTCGACCGAGGAAGGG ETPGTSTEPSEGSAPGTACCTCCGAGTCAGCTACCCCGGAGTCCGGTCCTGG STEPSEGSAPGTSESATCACCTCCACCGAACCATCGGAGGGCAGCGCCCCTG PESGPGTSESATPESGPGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAGAG GSPAGSPTSTEEGTSESGGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCC ATPESGPGSEPATSGSETGGTACTAGTACTGAACCATCCGAGGGGTCAGCTC TPGTSESATPESGPGTSCAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGA TEPSEGSAPGTSTEPSECCGGGCTCAGAGCCCGCCACGAGTGGCAGTGAAAC GSAPGTSTEPSEGSAPGTCCAGGCTCAGAACCCGCCACTAGTGGGTCAGAGA TSTEPSEGSAPGTSTEPCTCCAGGCAGCCCTGCCGGATCCCCTACGTCCACC SEGSAPGTSTEPSEGSAGAGGAGGGAACATCTGAGTCCGCAACACCCGAATC PGSPAGSPTSTEEGTSTCGGTCCAGGCACCTCCACGGAACCTAGTGAAGGCT EPSEGSAPGTSESATPECGGCACCAGGTACAAGCACCGAACCTAGCGAGGGC SGPGSEPATSGSETPGTAGCGCTCCCGGCAGCCCTGCCGGCAGCCCAACCTC SESATPESGPGSEPATSAACTGAGGAGGGCACCAGTACTGAGCCCAGCGAGG GSETPGTSESATPESGPGATCAGCACCTGGCACCAGCACCGAACCTAGCGAG GTSTEPSEGSAPGTSESGGGAGCGCCCCTGGGACTAGCGAGTCAGCTACACC ATPESGPGSPAGSPTSTAGAGAGCGGGCCTGGAACTTCTACCGAACCCAGTG EEGSPAGSPTSTEEGSPAGGGATCCGCTCCAGGCACCTCCGAATCCGCAACC AGSPTSTEEGTSESATPCCCGAATCCGGACCTGGCTCAGAGCCCGCCACCAG ESGPGTSTEPSEGSAPGCGGGAGCGAAACCCCTGGCACATCCACCGAGCCTA TSESATPESGPGSEPATGCGAAGGGTCCGCACCCGGCACCAGTACAGAGCCT SGSETPGTSESATPESGAGCGAGGGATCAGCACCTGGCACCAGTGAATCTGC PGSEPATSGSETPGTSETACACCAGAGAGCGGCCCTGGAACCTCCGAGTCCG SATPESGPGTSTEPSEGCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGC SAPGSPAGSPTSTEEGTTCCCCCACCTCAACAGAAGAGGGGACAAGCGAAAG SESATPESGPGSEPATSCGCTACGCCTGAGAGTGGCCCTGGCTCTGAGCCAG GSETPGTSESATPESGPCCACCTCCGGCTCTGAAACCCCTGGCACTAGTGAG GSPAGSPTSTEEGSPAGTCTGCCACGCCTGAGTCCGGACCCGGGACCTCTAC SPTSTEEGTSTEPSEGSTGAGCCCTCGGAGGGGAGCGCTCCTGGCACGAGTA APGTSESATPESGPGTSCAGAACCTTCCGAAGGAAGTGCACCGGGCACAAGC ESATPESGPGTSESATPACCGAGCCTTCCGAAGGCTCTGCTCCCGGAACCTC ESGPGSEPATSGSETPGTACCGAACCCTCTGAAGGGTCTGCACCCGGCACGA SEPATSGSETPGSPAGSGCACCGAACCCAGCGAAGGGTCAGCGCCTGGGACC PTSTEEGTSTEPSEGSATCAACAGAGCCCTCGGAAGGATCAGCGCCTGGAAG PGTSTEPSEGSAPGSEPCCCTGCAGGGAGTCCAACTTCCACGGAAGAAGGAA ATSGSETPGTSESATPECGTCTACAGAGCCATCAGAGGGGTCCGCACCAGGT SGPGTSTEPSEGSAPGSACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGG SSGTCCGAACCTGCCACCTCCGGCTCTGAAACTCCAG GGACCTCCGAATCTGCCACACCCGAGAGCGGCCCTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAGCACCGAGCCATCCGAGGGATCCGCC CCAGGCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCGCTGGCTCCCCCACCAGTACCG AGGAGGGGTCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCTGCCGGCTCCCCCACAAGTAC CGAAGAGGGCACAAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAGCACAGAGCCTTCCGAGGGG TCCGCACCAGGTACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTCCGGGT CTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCCACTTCCGG CTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACAGAGCCCTCT GAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCA CACCCGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCGC CACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGAGGAGGGATCTCCTGCTGGC TCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGCCATCCGAGGGATCAGCTCCCGGCACCTCAGAGT CGGCAACCCCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGA ATCAGCAACACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGATCAGAA CCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCACTGAGGAGGGCACCA GCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGTTCAGCACCCGGCTC TGAGCCGGCCACAAGTGGCAGTGAGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGC ACTAGTACCGAGCCCTCTGAAGGCAGTGCGCCAGGTTCGTCTTCATAA FIX-/FXI/- MQRVNMIMAESPGLITI 618atgcagcgcgtgaacatgatcatggcagaatcacc 619 XTEN_AE864 CLLGYLLSAECTVFLDHaggcctcatcaccatctgccttttaggatatctac  ENANKILNRPKRYNSGKtcagtgctgaatgtacagtttttcttgatcatgaa LEEFVQGNLERECMEEKaacgccaacaaaattctgaatcggccaaagaggta CSFEEAREVFENTERTTtaattcaggtaaattggaagagtttgttcaaggga EFWKQYVDGDQCESNPCaccttgagagagaatgtatggaagaaaagtgtagt LNGGSCKDDINSYECWCtttgaagaagcacgagaagtttttgaaaacactga PFGFEGKNCELDVTCNIaagaacaactgaattttggaagcagtatgttgatg KNGRCEQFCKNSADNKVgagatcagtgtgagtccaatccatgtttaaatggc VCSCTEGYRLAENQKSCggcagttgcaaggatgacattaattcctatgaatg EPAVPFPCGRVSVSQTSttggtgtccctttggatttgaaggaaagaactgtg KLTRAETVFPDVDYVNSaattagatgtaacatgtaacattaagaatggcaga TEAETILDNITQSTQSFtgcgagcagttttgtaaaaatagtgctgataacaa NDFTRVVGGEDAKPGQFggtggtttgctcctgtactgagggatatcgacttg PWQVVLNGKVDAFCGGScagaaaaccagaagtcctgtgaaccagcagtgcca IVNEKWIVTAAHCVETGtttccatgtggaagagtttctgtttcacaaacttc VKITVVAGEHNIEETEHtaagctcacccgtgctgagactgtttttcctgatg TEQKRNVIRIIPHHNYNtggactatgtaaattctactgaagctgaaaccatt AAINKYNHDIALLELDEttggataacatcactcaaagcacccaatcatttaa PLVLNSYVTPICIADKEtgacttcactcgggttgttggtggagaagatgcca YTNIFLKFGSGYVSGWGaaccaggtcaattcccttggcaggttgttttgaat RVFHKGRSALVLQYLRVggtaaagttgatgcattctgtggaggctctatcgt PLVDRATCLRSTKFTIYtaatgaaaaatggattgtaactgctgcccactgtg NNMFCAGFHEGGRDSCQttgaaactggtgttaaaattacagttgtcgcaggt GDSGGPHVTEVEGTSFLgaacataatattgaggagacagaacatacagagca TGIISWGEECAMKGKYGaaagcgaaatgtgattcgaattattcctcaccaca IYTKVSRYVNWIKEKTKactacaatgcagctattaataagtacaaccatgac LTGPEQTSKLTRAETVFattgcccttctggaactggacgaacccttagtgct PGSPAGSPTSTEEGTSEaaacagctacgttacacctatttgcattgctgaca SATPESGPGTSTEPSEGaggaatacacgaacatcttcctcaaatttggatct SAPGSPAGSPTSTEEGTggctatgtaagtggctggggaagagtGttccacaa STEPSEGSAPGTSTEPSagggagatcagctttagttcttcagtaccttagag EGSAPGTSESATPESGPttccacttgttgaccgagccacatgtctAcgatct GSEPATSGSETPGSEPAacaaagttcaccatctataacaacatgttctgtgc TSGSETPGSPAGSPTSTtggcttccatgaaggaggtagagattcatgtcaag EEGTSESATPESGPGTSgagatagtgggggaccccatgttactgaagtggaa TEPSEGSAPGTSTEPSEgggaccagtttcttaactggaattattagctgggg GSAPGSPAGSPTSTEEGtgaagagtgtgcaatgaaaggcaaatatggaatat TSTEPSEGSAPGTSTEPataccaaggtatcccggtatgtcaactggattaag SEGSAPGTSESATPESGgaaaaaacaaagctcactGGCCCAGAAcaaacAtc PGTSTEPSEGSAPGTSEtaagctAacGcgtgcGgagacAgtAtttccaGGTT SATPESGPGSEPATSGSCTCCAGCCGGGTCCCCAACTTCGACCGAGGAAGGG ETPGTSTEPSEGSAPGTACCTCCGAGTCAGCTACCCCGGAGTCCGGTCCTGG STEPSEGSAPGTSESATCACCTCCACCGAACCATCGGAGGGCAGCGCCCCTG PESGPGTSESATPESGPGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAGAG GSPAGSPTSTEEGTSESGGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCC ATPESGPGSEPATSGSETGGTACTAGTACTGAACCATCCGAGGGGTCAGCTC TPGTSESATPESGPGTSCAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGA TEPSEGSAPGTSTEPSECCGGGCTCAGAGCCCGCCACGAGTGGCAGTGAAAC GSAPGTSTEPSEGSAPGTCCAGGCTCAGAACCCGCCACTAGTGGGTCAGAGA TSTEPSEGSAPGTSTEPCTCCAGGCAGCCCTGCCGGATCCCCTACGTCCACC SEGSAPGTSTEPSEGSAGAGGAGGGAACATCTGAGTCCGCAACACCCGAATC PGSPAGSPTSTEEGTSTCGGTCCAGGCACCTCCACGGAACCTAGTGAAGGCT EPSEGSAPGTSESATPECGGCACCAGGTACAAGCACCGAACCTAGCGAGGGC SGPGSEPATSGSETPGTAGCGCTCCCGGCAGCCCTGCCGGCAGCCCAACCTC SESATPESGPGSEPATSAACTGAGGAGGGCACCAGTACTGAGCCCAGCGAGG GSETPGTSESATPESGPGATCAGCACCTGGCACCAGCACCGAACCTAGCGAG GTSTEPSEGSAPGTSESGGGAGCGCCCCTGGGACTAGCGAGTCAGCTACACC ATPESGPGSPAGSPTSTAGAGAGCGGGCCTGGAACTTCTACCGAACCCAGTG EEGSPAGSPTSTEEGSPAGGGATCCGCTCCAGGCACCTCCGAATCCGCAACC AGSPTSTEEGTSESATPCCCGAATCCGGACCTGGCTCAGAGCCCGCCACCAG ESGPGTSTEPSEGSAPGCGGGAGCGAAACCCCTGGCACATCCACCGAGCCTA TSESATPESGPGSEPATGCGAAGGGTCCGCACCCGGCACCAGTACAGAGCCT SGSETPGTSESATPESGAGCGAGGGATCAGCACCTGGCACCAGTGAATCTGC PGSEPATSGSETPGTSETACACCAGAGAGCGGCCCTGGAACCTCCGAGTCCG SATPESGPGTSTEPSEGCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGC SAPGSPAGSPTSTEEGTTCCCCCACCTCAACAGAAGAGGGGACAAGCGAAAG SESATPESGPGSEPATSCGCTACGCCTGAGAGTGGCCCTGGCTCTGAGCCAG GSETPGTSESATPESGPCCACCTCCGGCTCTGAAACCCCTGGCACTAGTGAG GSPAGSPTSTEEGSPAGTCTGCCACGCCTGAGTCCGGACCCGGGACCTCTAC SPTSTEEGTSTEPSEGSTGAGCCCTCGGAGGGGAGCGCTCCTGGCACGAGTA APGTSESATPESGPGTSCAGAACCTTCCGAAGGAAGTGCACCGGGCACAAGC ESATPESGPGTSESATPACCGAGCCTTCCGAAGGCTCTGCTCCCGGAACCTC ESGPGSEPATSGSETPGTACCGAACCCTCTGAAGGGTCTGCACCCGGCACGA SEPATSGSETPGSPAGSGCACCGAACCCAGCGAAGGGTCAGCGCCTGGGACC PTSTEEGTSTEPSEGSATCAACAGAGCCCTCGGAAGGATCAGCGCCTGGAAG PGTSTEPSEGSAPGSEPCCCTGCAGGGAGTCCAACTTCCACGGAAGAAGGAA ATSGSETPGTSESATPECGTCTACAGAGCCATCAGAGGGGTCCGCACCAGGT SGPGTSTEPSEGSAPGSACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGG SSGTCCGAACCTGCCACCTCCGGCTCTGAAACTCCAG GGACCTCCGAATCTGCCACACCCGAGAGCGGCCCTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAGCACCGAGCCATCCGAGGGATCCGCC CCAGGCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCGCTGGCTCCCCCACCAGTACCG AGGAGGGGTCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCTGCCGGCTCCCCCACAAGTAC CGAAGAGGGCACAAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAGCACAGAGCCTTCCGAGGGG TCCGCACCAGGTACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCACCTCCGGGT CTGAGACACCCGGGACTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCCACTTCCGG CTCCGAAACTCCCGGCACAAGCGAGAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACAGAGCCCTCT GAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCA CACCCGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCGC CACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCCACTGAGGAGGGATCTCCTGCTGGC TCTCCAACATCTACTGAGGAAGGTACCTCAACCGAGCCATCCGAGGGATCAGCTCCCGGCACCTCAGAGT CGGCAACCCCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACACCAGAGTCCGGTCCCGGGACTTCAGA ATCAGCAACACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACACCAGGATCAGAA CCTGCTACCTCAGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCACTGAGGAGGGCACCA GCACAGAACCAAGCGAGGGCTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGTTCAGCACCCGGCTC TGAGCCGGCCACAAGTGGCAGTGAGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGC ACTAGTACCGAGCCCTCTGAAGGCAGTGCGCCAGGTTCGTCTTCATAA

Example 25: Expression of FVII-XTEN and FIX-XTEN

Transient Transfection of Mammalian Cells

Mammalian cells, including CHO-K1, BHK, COS-7, and HEK293, were found toexpress FVII-XTEN or FIX-XTEN using different XTEN lengths whentransfected. The following are details for methods used to express thevarious FVII-XTEN and FIX-XTEN fusion protein constructs by transienttransfection.

HEK293 cells were plated the day before transfection, 1×10⁵ per well in12-well plate in 1 ml medium containing 10% FBS, 1×Pen/Strep, and 5mg/ml vitamin K. For transfection the day after plating the cells,plasmid DNA (0.6 μg) diluted in OptiMEM (total 25 μl) was mixed withdiluted FuGENE6 (2.1 μl FuGENE6 in 22.9 μl OptiMEM) and incubated for 30min at room temperature before adding to the cells. On day 3 or 4 aftertransfection the culture medium was collected, centrifuged at 500×g for5 min at room temperature, and then the supernatant filtered using 0.2μm filter before testing for expression of FVII-XTEN or FIX-XTEN inELISA and performance in a clotting assay (PT for FVII activity and aPTTfor FIX activity). The results are presented in Table 26.

It should be noted that the titer measured for FVII-XTEN by PT assay(active FVII protein) was higher than the titer measured by ELISA (totalFVII protein), and while the exact cause for this remained to beclarified, it could be due to (1) underestimation of FVII in the contextof FVII-XTEN due to epitope shielding by XTEN, (2) overestimation ofclotting activity by the PT assay, or a combination of both (1) and (2).It should also be noted that the titer measured for FIX by aPTT assay(active FIX protein) was significantly lower than the titer measured byELISA (total FIX protein), only about 20%, due to unknown reasons, butone of which could be insufficient propeptide processing, a phenomenonthat has been reported for recombinant FIX produced in CHO or othermammalian cells. The titer of FIX-XTEN by aPTT was even lowerproportionally than ELISA compared to FIX alone, suggesting the activityof FIX could be reduced by fusing to XTEN, an assumption confirmed byanalyzing the activity and ELISA titers of protein after TEV treatmentfor materials produced from cells transfected with plasmids constructsencoding a FVII-XTEN but with TEV cleavage site inserted in between.

TABLE 26 Expression of FVII, FVII-XTEN_AE864, FIX, and FIX-XTEN_AE864FVII FVII-XTEN FIX FIX-XTEN ELISA Clotting ELISA Clotting ELISA ClottingELISA Clotting ng/ml 391.0 397.7 34.8 176.6 309.8 60.0 13.7 0.4

Generation of CHO-K1 Stable Pools and Cell Lines that Produce FVII-XTEN

Cells: CHO-K1 cells purchased from ATCC (Cat. CCL-61, Lot 58078551) wererevived in Complete Medium (F-12K, 10% FBS and 1×P/S, Appendix 1) andpassaged for four generations before multiple vials were frozen in theComplete Medium with 5% DMSO. One vial was revived in medium similar tothe Complete Medium but with 5% FBS and passaged one more time beforetransfection.

Generation of stable pool: Construction of plasmids pBC0014, pBC0016,and pBC0018 encoding FVII-AE864, FVII-AE864, and FVII-AE288,respectively, has been described in the Examples above. Two of theplasmids pBC0016 and pBC0018 also carry UCOE. The plasmids were firstlinearized with PvuII and then transfected with FuGENE6 transfectionreagent into separate T25 flasks of CHO-K1 cells from above, 3.6 μgplasmid DNA for 6.5×10⁵ cells per flask. Two days later the cells weretransferred to T75 and cultured in Selection Medium (Complete Mediumwith 10 μg/ml puromycin and 5 μg/ml vitamin K). The flasks were changedto fresh Selection Medium every 2-3 days. Two weeks after transfection,cells from T75 flasks were frozen as stable pool.

Selection of clones: For primary screening, frozen stable pool cellswere revived and seeded in 6 96-well plates at a target density of 0.5cell/well. About 1 week after seeding spent medium from wells withsingle cell cluster as observed under microscope were tested forexpression of FVII by ELISA. The number of clones tested in the primaryscreening by the ELISA was: 154 for pBC0014, 210 for pBC0016, and 135for pBC0018. Significant numbers of clones expressed no ornon-detectable levels of FVII (FIG. 9, black bars, expressed as ng/ml),but 15-20% of the clones expressed FVII of 3-8 fold higher, these cloneswere then selected for further screening and selection, 20 for pBC0014,30 for pBC0016, and 20 for pBC0018. The size of the cell clusters inthese wells was scored 1-10 with 1 being the smallest cluster and 10 thelargest cluster; the results are shown as gray bars in FIG. 9. Thedistribution of the cell cluster size of these clones was similar tothat of all the clones for the same variant, suggesting they wereselected not just because they were the fastest growers.

For additional rounds of screening, normalized numbers of cells wereseeded in multi-well plates. Spent medium were harvested 2-3 days afterseeding and were tested for FVII concentration by ELISA and clottingactivity by PT; Cells were also harvested from the plates and countedusing Vi-Cell. Clones were ranked by (1) FVII titers according to ELISAand clotting; (2) Ratios of ELISA titer/cell count and clottingtiter/cell count; and (3) Integrity and homogeneity of products producedby the clones as measured by Western blots. Selection of clones for eachof the construct pBC0014, pBC0016, and pBC0018 was described separatelyin the following.

pBC0014: For the second round of screening, cells in 96-well plates forthe top 20 clones selected from primary screening were first expanded inT25 flasks and then seeded in duplicate 24-well plates, one cultured for2 days and the other one for 3 days. Spent medium were collected fromthe plates for FVII ELISA and cells harvested and counted by Vi-Cell.Fourteen clones were selected according to titers by ELISA and clotting,ELISA titer/cell and clotting titer/cell count ratios and furtherscreened. Frozen vials were prepared for 9 clones, 1F10, 2F7, 6H4, 1A3,6F10, 5C2, 5F1, 3H2, 4C8. Out of the 14 clones, 1F10, 1F4, 2F7, 4C8,6H4, and 6G1 were again screened and ranked according to titers by ELISAand clotting, ratios of ELISA titer/cell count and clotting titer/cellcount, and product integrity and homogeneity by Western blot (FIGS.10-12). Clone 6G1 expressed a product that is significantly smaller thanthe full-length product (FIG. 12) and was discarded. Additional frozenvials were prepared for clones 1F10, 2F7, 6H4, and 4C8. Clone 4C8 wastested for production of FVII-AE864 in roller bottles.

pBC0016: For the second round of screening, cells in 96-well plates forthe top 30 clones selected from primary screening were transferred to12-well plates and then T25 and ranked by titers according to ELISA andclotting assays, ratios of ELISA titer/cell count and clottingtiter/cell count. For the third round of screening, fifteen clonesincluding 1D4, 1G2, 1G6, 2C11, 2H6, 3A2, 3B1, 3C7, 3F2, 3H1, 3H6, 3H10,4G8, 5E12, 6F11 were tested, ranked according to above criteria plusWestern blot (FIG. 13) and frozen cells were prepared for all of the 15clones, except 3H6. Eight clones including 1G2, 2C11, 3B1, 3C7, 3F2,3H10, 4G8, 5E12 were selected as the top clones and additional frozenvials were prepared for them. Clone 3H10 was selected for scale-upproduction in roller bottles.

pBC0018: For the second round of screening, cells in 96-well plates forthe top 20 clones selected from primary screening were first expanded inT25 flasks and then seeded in 24-well plates. Spent medium werecollected from the plates for FVII ELISA and cells harvested and countedby Vi-Cell. Twelve clones were selected according to titers by ELISA andclotting, ELISA titer/cell and clotting titer/cell count ratios andfurther screened. Frozen vials were prepared for 9 clones, 2C3, 2D5,3B2, 3B10, 3G2, 3G12, 5A12, 6A3, and 6E7. Out of the 9 clones, 2D5, 3B2,3G2, 3G12, 5A12, 6A3, and 6E7 were again screened and ranked accordingto titers by ELISA and clotting, ratios of ELISA titer/cell count andclotting titer/cell count, and product integrity and homogeneity byWestern blot (FIG. 14). Clone 3B2 expressed products that displayedmultiple bands on Western blot and it was discarded. Additional frozenvials were prepared for clones 2D5, 3G2, 3G12, 5A12, 6A3, 6E7. Clones3G12 and 6E7 were used for production of FVII-AE288 in roller bottles.

Production of FVII-XTEN Secreted in Cell Culture Medium in RollerBottles

CHO-K1 cell stable pools or clones were expanded in T175, 35 mlSelection Medium per flask. Cells were harvested from by trypsinizationand used to seed roller bottles (1700 cm² surface area per bottle) onDay 0, 300 ml Selection Medium with cells from 1-2 T175 flasks for everyroller bottle. The spent/conditioned medium was removed on Day 3 (or 4)and refilled with 300 ml fresh Selection Medium. On Day 5 (or 6) thespent/conditioned medium was removed and discarded (or harvested if XTENfusion proteins in this medium can be purified) and 300 ml TransitionMedium (UltraCHO containing 1% FBS, 0.1% Ex-Cyte, 5 mg/ml vitamin K, and1×Pen/Step) was added to each roller bottle. On Day 7 (or 8) the spentmedium was removed and discarded (or harvested if XTEN fusion proteinscan be purified from this medium) and Expression Medium (OptiMEMcontaining 0.1% Ex-Cyte, 1% ITS-A, 5 mg/ml vitamin K, and 1×Pen/Strep)was added, 300 ml per bottle or other volumes depending on results fromoptimization. Conditioned medium could be harvested once everyday, oronce every 2, or 3, or 4 days depending on product titer and qualitydesired. To harvest, the conditioned medium was poured into centrifugebottles, and fresh Expression Medium was added, 300 ml per bottle orother volumes depending on results from optimization. This production ofharvesting spent medium and refilling with fresh medium could last for2-4 weeks until titer or/and product quality are considered too low,when the roller bottles are terminated. The conditioned medium was thencentrifuged, 3500 rpm in a bench-top centrifuge, for 10 min, at 4-8° C.The supernatant was then filtered using a 0.2 mm filter. The filtratewas either processed immediately or was stored in −80° C. freezer beforeprocessing by tangential flow filtration (TFF) for purification.

Example 26: Purification and Characterization of FVII-XTEN Constructs

Concentration and Buffer Exchange of FVII-XTEN_AE864 by Tangential FlowFiltration and Diafiltration

Supernatant batches S279, S281, S282 and S287, totaling 10.7 L involume, from stable CHO cells lines expressing FVII-AE864 (AC404) werefiltered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssurecapsule. They were subsequently concentrated approximately 20 fold bytangential flow filtration using a Millipore Pellicon 2 Mini cartridgewith a 30,000 Da MWCO. Using the same tangential flow filtrationcartridge the sample was diafiltered with 10 mM tris pH 7.5, 1 mM EDTAwith 5 volumes worth of buffer exchange. Samples were divided into 50 mlaliquots and frozen at −80° C. No FVII activity was detectable in thepermeate fractions from the filtration and ˜75% recovery of the activitywas seen in the concentrated, buffer exchanged material.

Purification of FVII-XTEN_AE864 by BaSO₄ Adsorption

FVII-AE864 (AC404) containing supernatant was concentrated and bufferexchanged into 10 mM tris pH 7.5, 1 mM EDTA. Subsequently, 5 ml of thissample was diluted 10 fold in PBS, additional NaCl was added to 50 mM,and then BaSO₄ was added to 20 mg/ml. The sample was bound on a nutatorat room temperature for 30 minutes. The sample was then centrifuged at3000 rpm for 5 minutes to pellet the BaSO₄. The supernatant wasdiscarded and the pellet resuspended in 5 ml if 200 mM sodium acetateand nutated for 30 minutes at room temperature. This was repeated twomore times. After the third wash the pellet was resuspended in 0.8 ml of100 mM trisodium citrate pH 7.0 and nutated for 30 minutes at roomtemperature. This was repeated once. A Bradford assay was performed todetermine the total amount of protein in the sample and FVII activitywas assayed in a PT based factor assay with Innovin as the activatingthromboplastin (Table 27). The ratio of activity to total proteindemonstrated a net purification of ˜12 fold from this purification step.

TABLE 27 Purification Table of FVII-AE864 by BaSO4 Absorption TotalTotal Volume Activity Protein Specific Purity Fraction (ml) (U) (ug)Activity Purification (%) Feed 5 17.3  6300 0.003 1.0 0.1% FT 1 5 4.04687 0.001 0.3 0.0% Wash 1 5 NA 57 NA NA NA Wash 2 5 NA 8 NA NA NA Wash3 5 NA 8 NA NA NA Elution 1 0.8 3.2 85 0.038 13.8 1.9% Elution 2 0.8 0.412 0.030 11.0 1.5%

Purification of FVII-XTEN_AE864 by aGla Affinity Chromatography

A monoclonal antibody (clone ID CaFVII-22) which binds the GLA domain ofFVII in a calcium dependent manner was coupled to Ultralink beads fromPierce. Coupling was performed by adding 10 mg of antibody in PBS to1.25 of resin and bringing the final volume to 15 ml with couplingbuffer (100 mM MOPS, 700 mM sodium citrate, pH 8.0). This produced 10 mlof resin slurry and a 1 mg to 1 ml ratio of antibody mass to bead slurryvolume. The slurry was incubated for 2 hours at room temperature andthen the beads were washed with coupling buffer. A BCA assay indicated˜70% of the antibody was coupled to the beads. The beads are thenquenched with 1M tris pH 8.0 for 2 hours at room temperature. The beadswere equilibrated into 10 mM tris pH 7.5 and 10 mM CaCl₂ and 5.5 ml ofbeads was mixed with 50 ml of concentrated, buffer exchanged FVII-AE864(AC404) supernatant in 10 mM tris pH 7.5 and ˜10 mM CaCl₂. The samplewas incubated at 4° C. overnight on a nutator to bind the FVII-XTEN tothe resin. The following day the beads were washed three times with 45ml 10 mM tris, 500 mM NaCl, 10 mM CaCl₂, pH 7.5 and then eluted with 20ml of 10 mM tris, 100 mM EDTA, pH 7.5. SDS-PAGE analysis indicates thatthe purity is in excess of 90% (FIG. 15).

Activation of FVII-XTEN_AE864 and FVII-XTEN_AE288

Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398) wereactivated to FVIIa-AE864 and FVIIa-AE288 by addition of FXa. TheFVII-XTEN proteins were buffer exchanged into 10 mM Tris, 10 mM CaCl₂,pH7.5 via repeat rounds of concentration in an Amicon ultra 10,000 DaMWCO concentrator and subsequent dilution. The final volume was 1 ml at˜0.4 mg/ml. FXa from Novagen was added to a final concentration of 10units/ml and the sample incubated overnight at 4° C. Reducing SDS-PAGEindicated a complete conversion of FVII-XTEN proteins to FVIIa-XTENproteins by the downward shift in the top band with DTT compared to thenon-reduced sample which represents the loss of the light chain from themolecule, which can only occur upon activation (FIG. 16). Additionally,the light chain can be seen appearing lower on the gel and running atthe same position as the light chain of control FVIIa, furtherconfirming the transition of the FVII domain from FVII to FVIIa. Undersimilar buffer conditions FVII-XTEN fusions are activated to FVIIa-XTENby the addition of thrombin, FIXa, FXIIa or any other protease capableof selectively cutting the peptide bond between R152 and 1153.

Autoactivation of FVII-XTEN_AE864 and FVII-XTEN_AE288

Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398) wereactivated to FVIIa-AE864 and FVIIa-AE288 by incubating the sample at 4°C. for 1 week. The FVII-XTEN proteins were buffer exchanged into 10 mMTris, 10 mM CaCl₂, pH7.5 via repeat rounds of concentration in an Amiconultra 10,000 Da MWCO concentrator and subsequent dilution. After theincubation the protein was assayed by SDS-PAGE and the top band displaysthe characteristic downward shift in the top band with DTT compared tothe non-reduced sample which represents the loss of the light chain fromthe molecule, which can only occur upon activation (FIG. 17).Additionally, the light chain can bee seen appearing lower on the gel atthe same point as the two lots of FXa activated material, furthercorroborating the conclusion that the proteins auto-activated toFVIIa-XTEN.

Purification of FVII-XTEN_AE864 by Anion Exchange Chromatography

A sample of FVII-AE864 (AC404) containing supernatant was concentratedand buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA and then adjustedto a final concentration of ˜5 mM CaCl₂ with the addition of 1M CaCl₂.The sample was loaded onto a 2 ml macrocap Q column equilibrated on anAkta chromatography system. The protein was eluted with a lineargradient of 0-100% buffer B over 20 column volumes. Buffer A wascomprised of 20 mM MES, 5 mM CaCl₂ pH 6.0 and buffer B was comprised of20 mM MES, 5 mM CaCl₂ pH 6.0 and 500 mM NaCl. Fractions were assayed forFVII activity using a PT based factor assay with Innovin as theactivating thromboplastin. A single tight peak of activity was seeneluting between 47.9 and 52.4 ml, or 23.2 to 27.8 mS/cm (FIG. 19). ABradford assay was performed to determine the total amount of protein inthe load and elution fractions. The ratio of the activity to the totalprotein demonstrated an ˜5 fold net purification from the column.

Purification of FVII-XTEN_AE864 by Hydrophobic InteractionChromatography

A sample of FVII-AE864 (AC404) containing supernatant was concentratedand buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA and then adjustedto a final concentration of ˜5 mM CaCl₂ with the addition of 1M CaCl₂.The sample was loaded onto a 2 ml toyopearl phenyl column equilibratedon an Akta chromatography system. The protein was eluted with a lineargradient of 0-100% buffer B over 20 column volumes. Buffer A wascomprised of 10 mM Tris, 5 mM CaCl2, 3M NaCl, pH 7.5 and buffer B wascomprised of 10 mM Tris, 5 mM CaCl₂, pH 7.5. Fractions were assayed forFVII activity using a PT based factor assay with Innovin as theactivating thromboplastin. A single peak of activity was seen elutingbetween 1M and 2M NaCl (FIG. 20). A Bradford assay was performed todetermine the total amount of protein in the load and elution fractions.The ratio of the activity to the total protein demonstrated an ˜2 foldnet purification from the column.

Removal of Aggregated protein from monomeric FVII-AE864 with AnionExchange Chromatography

Affinity purified FVII-AE864 (AC404) was loaded was adjusted to pH 6.0by addition of 200 mM MES, 210 mM CaCl2 pH 6.0 at a ratio of 1 ml bufferto 10 ml sample. Using an Akta FPLC system the sample was purified usinga 2 ml macrocap Q column. The column was equilibrated into buffer A (20mM MES, 1 mM CaCl2, pH 6.0) and the sample loaded. The sample was elutedusing a linear gradient of 30% to 80% buffer B (20 mM MES, 1 mM CaCl2,pH 6.0+500 mM NaCl) over 20 column volumes. The 215 nm chromatogramshowed two peaks in the elution profile (FIG. 21A). The fractionscorresponding to the early peak and the late peak were pooled andanalyzed via size exclusion chromatography (SEC) with 60 cm BioSep G4000column. The early peak contained a monodispersed population with acharacteristic hydrodynamic radius of a monomeric AE864 protein (10.1 nmor apparent MW of 1.9 MDa) (FIG. 21B). The late peak contained twopopulations, the smaller monomeric peak demonstrating the absence ofaggregates in the early peak. and an earlier eluting, larger peak at thevoid volume of the column (22 ml) characteristic of aggregated protein.

SEC Analysis of FVII-AE864 and FVII-AE288

FVII-AE864 (AC404) and FVII-AE288 (AC398) were purified by affinity andanion exchange chromatography and characterized. Size exclusionchromatography with 60 cm BioSep G4000 column indicated a monodispersedpopulation with a characteristic hydrodynamic radius for either amonomeric AE864 protein (10.1 nm or apparent MW of 1.9 MDa for anapparent molecular weight factor of 15.2) or a monomeric AE288 protein(8.2 nm or apparent MW of 650 kDa for an apparent molecular weightfactor of 9.0) (FIG. 18). Minimal aggregation was seen in either sample.SDS-PAGE showed a >90% pure protein with minimal host cell proteincontamination.

Lipidated Tissue Factor Initiated Clotting Activity Analysis ofFVII-AE864 and FVII-AE288

Activity was assayed by a PT based factor VII assay as follows: Astandard curve was prepared by diluting normal plasma ten fold with FVIIdeficient plasma and then conducting 4, 5 fold serial dilutions againwith factor VII deficient plasma. This created a standard curve withpoints at 100, 20, 4, 0.8 and 0.16 mUnits/ml of activity, where one unitof activity is defined as the amount of FVII activity in 1 ml of normalhuman plasma. A FVII-deficient plasma was also included to determine thebackground level of activity in the null plasma. The sample was preparedby adding FVII-XTEN to FVII deficient plasma at a ratio of 1:10 byvolume. The samples were incubated at 37 C in a molecular devices platereader spectrophotometer for 3 minutes at which point the clottingreaction was initiated by the addition of 2 volumes of thromboplastin(Dade Innovin, B4212-50) per one volume of sample. The turbidity wasmonitored at 405 nm for 5 minutes to create reaction profiles. The PTtime, or time to onset of clotting activity, was defined as the firsttime where OD405 nm increased by 0.06 over baseline. A log-linearstandard curve was created with the log of activity relating linearly tothe PT time. From this the activity of the sample in the plate well wasdetermined and then the activity in the sample determined by multiplyingby 11 to account for the dilution into the FVII deficient plasma. Basedupon quadruplicate measurements the activity of the FVII-AE864 (AC404)fusion was 30 Units/ml and the FVII-AE288 (AC398) was 15 U/ml.Additionally, this lapidated tissue factor activation of clotting isused to assay the activity of FVII-XTEN fusions in clotting assays withmore sophisticated readouts like thrombin generation assays, TEG assays,rotem assays and other in vitro/ex vivo which involve the detection ofclotting enzyme function by substrate turnover, mechanical clotformation or photo-optical clot detection.

Soluble Tissue Factor Initiated Clotting Activity Analysis of FVII-AE864and FVII-AE288

After activation of FVII-AE288 (AC398) to FVIIa-AE288 the activity wasmeasured by soluble tissue factor (sTF) induced clotting. This isperformed using the Stago STA-Clot FVIIa activity assays kit. Briefly,the samples were incubated with sTF, which binds and enhances FVIIaactivity, but does not convert FVII to FVIIa, The time to induce a clotin FVII null plasma was defined as the first time where OD405 nmincreased by 0.06 over baseline when monitored in the molecular devicesplate reader. This time is compared to a standard curve comprised ofknown FVIIa amounts added into FVII null plasma, and an activity numbercalculated. The FVIIa-AE288 sample contained an activity equivalent to112 U/ml of FVIIa activity. Additionally, this soluble tissue factoractivation of clotting is used to assay the activity of FVII-XTENfusions in clotting assays with more sophisticated readouts likethrombin generation assays, TEG assays, rotem assays and other invitro/ex vivo which involve the detection of clotting enzyme function bysubstrate turnover, mechanical clot formation or photo-optical clotdetection.

ELISA Based Concentration Determination of FVII-AE864 and FVII-AE288

FVII-XTEN fusion concentrations were determined using and ELISA assaywith an affinity purified polyclonal sheep anti-human FVII antibody,where an unmodified form of the antibody is used to capture the proteinand the HRP conjugated form was used to detect the protein. The captureantibody was coated at 4 C overnight on to a high binding 96 well assayplate (Corning 3690). The plate was blocked with 3% BSA in PBS for 1hour at room temperature. The plate was washed 6 times in PBST with aplate washer. Samples or standards, diluted in PBST, were then boundinto the appropriate wells for 2 hours at room temperature. The standardcurve ranged from 10 ng/ml to <1 μg/ml and was prepared by seriallydiluting commercial plasma derived FVII of a known concentration (AbcamCat #ab62386) in PBST. The plate was again washed 6 times with PBSTusing a plate washer. The FVII-XTEN was then detected using thedetection antibody which was bound for 1 hour at 37 C. The plate wasagain washed 6 times with PBST using a plate washer and washed onefurther time with water. Signal was then developed with TMB substrateand quantified by reading at 405 nm on a molecular devices plate readerspectrophotometer. A four parameter fit is then performed on thestandards and the concentration of the samples determined by comparisonto the standard curve.

Assessment of FVII-AE864 and FVII-AE288 Activity via Direct Turnover ofa Fluorogenic Substrate

FVII-XTEN fusion activity is determined by monitoring the cleavage of apeptide bond in the substrate D-FPR-6-amino-1-naphthalenesulfonamide(D-FPR-ANSN) where the DFPR moiety is a peptide chain linked to asecondary amine in the ANSH moiety. When the bond between the arginineresidue and the ANSH moiety is cleaved by the serine protease activityof the FVII catalytic domain the ANSH is released and becomes an intensefluorophore. FVII-XTEN activity is measured at enzyme concentrationsranging from 50 pM to 1 μM with substrate concentrations ranging from 50μM to 100 μM in 20 mM tris pH 8.0, 135 mM NaCl. By monitoring the changein ANSN fluorescence (excitation 352 nm, emission 470 nm) over time theactivity of the FVIIa catalytic domain can be determined. This activitycan be compared to a standard curve derived from FVIIa to determine theamount of FVIIa equivalents in the sample, or kinetic properties such askcat and Km for can be determined.

Assessment of FVII-AE864 and FVII-AE288 Activity Via a FXa CoupledChromogenic Substrate Assay

When complexed to Tissue Factor (TF), in presence of phospholipids andCalcium, FVII and FVII-XTEN activate factor X to factor Xa. BiophenFactor VII is a chromogenic assay for testing factor VII activity.Factor VII forms an enzymatic complex with Tissue Factor, provided byrabbit Thromboplastin. It then activates factor X, present in the assayat a constant concentration and in excess, to factor Xa. Theconcentration of FXa is exactly measured by its activity on a specificfactor Xa chromogenic substrate (SXa-11). Factor Xa cleaves thesubstrate and generates pNA. The amount of pNA generated is directlyproportional to the factor Xa activity. Finally, there is a directrelationship between the amount of factor VII activity in the assayedsample and the factor Xa activity generated, measured by the amount ofpNA released, determined by color development at 405 nm. By comparingthe signal from an unknown sample to the signal from a standard curve ofknow FVII activity, it is possible to calculate the amount of FVIIactivity in an unknown sample.

Example 27: ELISA Assays for FIX-XTEN Concentration Determination

FIX-XTEN concentrations were determined using and ELISA assay with aspecific matched pair of antibodies, where the detection antibody wasconjugated to HRP to simplify detection (Affinity Biologicals cat#FIX-EIA). The capture antibody was coated at 4 C overnight on to a highbinding 96 well assay plate (Corning 3690). The plate was blocked with3% BSA in PBS for 1 hour at room temperature. The plate was washed 6times in PBST with a plate washer. Samples or standards, diluted inPBST, were then bound into the appropriate wells for 2 hours at roomtemperature. The standard curve ranged from 25 ng/ml to <1 pg/ml and wasprepared by serially diluting commercial plasma derived FIX of a knownconcentration (Abcam Cat #ab62544) in PBST. The plate was again washed 6times with PBST using a plate washer. The FIX was then detected usingthe detection antibody which was bound for 1 hour at 37 C. The plate wasagain washed 6 times with PBST using a plate washer and washed onefurther time with water. Signal was then developed with TMB substrateand quantified by reading at 405 nm on a molecular devices plate readerspectrophotometer. A four parameter fit is then performed on thestandards and the concentration of the samples determined by comparisonto the standard curve.

Example 28: aPTT Based Assays for FIX-XTEN Activity Determination

FIX-XTEN would act to replace FIX in the intrinsic or contact activatedcoagulation pathway. The activity of this coagulation pathway isassessed using an activated partial thromboplastin time assay (aPTT).FIX activity specifically was measured as follows, a standard curve wasprepared by diluting normal control plasma (Pacific Hemostasis cat#100595) two fold with FIX deficient plasma (cat #100900) and thenconducting 6, 4 fold serial dilutions again with factor IX deficientplasma. This created a standard curve with points at 500, 130, 31, 7.8,2.0, 0.5 and 0.1 mUnits/ml of activity, where one unit of activity isdefined as the amount of FIX activity in 1 ml of normal human plasma. AFIX-deficient plasma was also included to determine the background levelof activity in the null plasma. The sample was prepared by addingFIX-XTEN to FIX deficient plasma at a ratio of 1:10 by volume. Thesamples were tested using an aPTT assay as follows. The samples wereincubated at 37 C in a molecular devices plate reader spectrophotometerfor 2 minutes at which point an equal volume of aPTT reagent (PacificHemostasis cat #100402) was added and an additional 3 minute 37 Cincubation performed. After the incubation the assay was activated byadding one volume of calcium chloride (Pacific Hemostasis cat #100304).The turbidity was monitored at 450 nm for 5 minutes to create reactionprofiles. The aPTT time, or time to onset of clotting activity, wasdefined as the first time where OD405 nm increased by 0.06 overbaseline. A log-linear standard curve was created with the log ofactivity relating linearly to the aPTT time. From this the activity ofthe sample in the plate well was determined and then the activity in thesample determined by multiplying by 11 to account for the dilution intothe FIX deficient plasma.

Example 29: FIX/cFXI/XTEN has Enhanced Activity Compared to FIX-XTEN

FIX (pCW0596), FIX-XTEN (pCW0597), FIX/cFXI1/XTEN (pCW0735),FIX/cFXI2/XTEN (pCW0736) and FIX/cFXI3/XTEN (pCW0737) were transientlyexpressed in CHO-K1 cells. Transient transfection supernatants wereconcentrated in 30,000 MWCO concentrators by approximately 15-fold. Theconcentration of the concentrated and unconcentrated samples wasdetermined by ELISA. The clotting activity of the concentrated sampleswas then determined using an aPTT based factor assay. For the XTENcontaining the activity was drastically altered by the presence any ofthe FXIc cleavage sites. In all three cases the presence of a FXIcleavage site enhanced the clotting activity by greater than 30-fold(see FIG. 22 and Table 28). The relatively consistent ELISA measurementindicates that this is an enhancement of the specific activity, ratherthan a change in titer. Additionally, the ratio of the activitymeasurement to the ELISA concentration for the FXI cleavage siteconstructs was now similar to the ratios for FIX, indicating that theFIX-FXIc-XTEN contained a FIX domain of similar properties to the FIXdomain expressed in the absence of XTEN.

TABLE 28 Activity of FIX/cFXI/XTEN Constructs ConcentrationConcentration by ELISA by Activity Fraction Construct (ng/ml) (ng/ml)Active FIX/cTEV/XTEN NA NA  4% FIX 197 30 15% FIX-XTEN 10 0  2%FIX/cFXI1/XTEN 23 7 30% FIX/cFXI2/XTEN 26 9 34% FIX/cFXI3/XTEN 29 11 40%

Example 30: Pharmacokinetic Analysis of CFXTEN Fusion Polypeptide inRats-FVII-XTEN_AE864

The pharmacokinetics of the CFXTEN FVII-XTEN_AE864, compared to FVIIalone, were tested in Sprague-Dawley. FVII-XTEN_AE864 and FVII wereadministered to female Sprague-Dawley rats (n=3) IV through a jugularvein catheter at 3 μg/rat. Blood samples (0.2 mL) were collected intoprechilled heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48,72 hour time points, and processed into plasma. Quantitation of the testarticles was performed by ELISA assay using an anti-FVII antibody forboth capture and detection. A non-compartmental analysis was performedin WinNonLin with all time points included in the fit to determine thePK parameters.

The pharmacokinetic results are summarized in Table 29 and FIG. 23. Thedata show XTEN can greatly extend the half-life of FVII as a CFXTENfusion protein as compared to FVII alone; FVII-XTEN has a half life ofapproximately 38 hours as compared to 1 hour for FVII. In additionFVII-XTEN was confined to the bloodstream, with a calculated volume ofdistribution of 50.8 mL in rats, indicating little extravasation intothe extracellular space.

TABLE 29 Half-life of FVII test articles in rats Test Article(Construct) T ½ (hrs) FVII-XTEN(AP315) 37.9 FVII (P318) 1.0

Example 31: Pharmacokinetic Analysis of CF XTEN Fusion Polypeptide inRats-FIX-XTEN_AE864

The pharmacokinetics of macrocap Q purified FIX-XTEN_AE864 were testedin Sprague-Dawley rats (n=3) and compared to unpurified FIX-XTEN,FIX-XTEN TEV cleaved (a preparation in which the XTEN is removed fromthe fusion protein by use of TEV protease), and commercially-availableFIX Benefix. Compounds were administered to female Sprague-Dawley ratsIV through jugular vein catheter at 3 μg/rat. Blood samples (0.2 mL)were collected into prechilled heparinized tubes at predose, 0.08, 0.5,1, 2, 4, 8, 24, 48, 72 hour time points, and processed into plasma.Quantitation of the test articles was performed by ELISA assay using ananti-FIX antibody for both capture and detection. A non-compartmentalanalysis was performed in WinNonLin with all time points included in thefit to determine the PK parameters.

The pharmacokinetic results are summarized in Table 30 and FIG. 24. Thedata show XTEN can greatly extend the half life of FIX as a CFXTENfusion protein as compared to either FIX-XTEN TEV cleaved or FIXBenefix; FIX-XTEN has a half life of 34.7 hours as compared to 4.6 hourfor FIX Benefix. In addition FIX-XTEN was confined to the bloodstreamwith a calculated volume of distribution of 38 mL in rats, indicatinglittle extravasation into the extracellular space.

TABLE 30 Half-life of FIX test articles in rats Test Article (Construct)T ½ (hrs) FIX-XTEN macro cap Q 34.7 (AP316a) FIX-XTEN (AP316) 33.1FIX-XTEN TEV (AP316b) 1.5 FIX Benefix 3.3

Example 32: Pharmacodynamic Evaluation of FVIIa-XTEN_AE864 in AnimalModels

The in vivo pharmacologic activity of FVIIa-XTEN constructs is assessedusing a variety of preclinical models of bleeding including but notlimited to those of hemophilia, surgery, trauma,thrombocytopenia/platelet dysfunction, clopidogrel/heparin-inducedbleeding and hydrodynamic injection. These models can be developed inmultiple species including mice, rat, rabbits, and dogs using methodsequivalent to those used and published for other FVIIa approaches.FVIIa-XTEN compositions are provided in an aqueous buffer compatiblewith in vivo administration (for example: phosphate-buffered saline orTris-buffered saline). The compositions are administered at appropriatedoses, dosing frequency, dosing schedule and route of administration asoptimized for the particular model. Efficacy determinations includemeasurement of FVIIa activity, prothrombin time (PT), activated partialprothrombin time (aPTT), bleeding time, whole blood clotting time(WBCT), thrombelastography (TEG or ROTEM), among others.

In one example of a PD model, FVIIa-XTEN and FVII are administered togenetically-deficient or experimentally-induced HemA or HemB mice. Atvarious time points post-administration, levels of FVIIa and FVIIa-XTENare measured by ELISA, activity of FVIIa and FVIIa-XTEN are measured bycommercially-available FVIIa activity kits and clotting time is measuredby PT assay. Overall, the results can indicate that the FVIIa-XTENconstructs may be more efficacious at inhibiting bleeding as compared toFVIIa and/or equivalent in potency to comparable dosage of FVIIa withless frequent or more convenient dosing intervals.

In a mouse bleeding challenge PD model FVIIa-XTEN and FVIIa areadministered to genetically-deficient or experimentally-induced HemA orHemB mice and effect on hemostatic challenge is measured. Hemostaticchallenge can include tail transaction challenge, hemarthropthychallenge, joint bleeding or saphenous vein challenge among others. Atvarious time points post-administration levels of FVII and FVIIa-XTENare measured by ELISA, activity of FVII and FVIIa-XTEN are measured bycommercially available FVIIa activity kit, bleeding time is measured andclotting time is measured by PT assay. Overall the results can indicatethat the VIIa-XTEN constructs may be more efficacious at inhibitingbleeding as compared to FVIIa and/or equivalent in potency to comparabledosage of FVIIa with less frequent or more convenient dosing intervals.

In a dog PD model, FVIIa-XTEN and FVII are administered togenetically-deficient hemophiliac dogs. At various time points postadministration, levels of FVIIa and FVIIa-XTEN are measured by ELISA,activity of FVIIa and FVIIa-XTEN are measured by commercially availableFVIIa activity kit and clotting time is measured by PT assay. Overallthe results can indicate that the FVIIa-XTEN constructs may be moreefficacious at inhibiting bleeding as compared to FVIIa and/orequivalent in potency to comparable dosage of FVIIa with less frequentor more convenient dosing.

In a dog bleeding challenge PD model FVIIa-XTEN and FVIIa areadministered to genetically deficient hemophiliac dogs and effect onhemostatic challenge is measured. Hemostatic challenge can includecuticle bleeding time among others. At various time points postadministration levels of FVII and FVIIa-XTEN are measured by ELISA,activity of FVII and FVIIa-XTEN are measured by commercially availableFVIIa activity kit, bleeding time is measured and clotting time ismeasured by PT assay. Overall the results can indicate that theVIIa-XTEN constructs may be more efficacious at inhibiting bleeding ascompared to FVIIa and/or equivalent in potency to comparable dosage ofFVIIa with less frequent or more convenient dosing intervals.

Additional preclinical models of bleeding include but are not limited tothose of hemophilia, surgery, trauma, thrombocytopenia/plateletdysfunction, clopidogrel/heparin-induced bleeding and hydrodynamicinjection. These models can developed in multiple species includingmice, rat, rabbits, and dogs using methods equivalent to those used andpublished for other FVIIa approaches. Overall the results can indicatethat the FVIIa-XTEN constructs may be more efficacious at inhibitingbleeding as compared to FVIIa and/or equivalent in potency to comparabledosage of FVIIa with less frequent or more convenient dosing intervals.

Example 33: Pharmacodynamic Evaluation of FIX-XTEN_AE864 in AnimalModels

The in vivo pharmacologic activity of FIX-XTEN constructs is assessedusing a variety of preclinical models of bleeding including, but notlimited to, those of hemophilia, surgery, trauma,thrombocytopenia/platelet dysfunction, clopidogrel/heparin-inducedbleeding and hydrodynamic injection. These models can be developed inmultiple species including mice, rat, rabbits, and dogs using methodsequivalent to those used and published for other FIX approaches.FIX-XTEN compositions are provided in an aqueous buffer compatible within vivo administration (for example: phosphate-buffered saline orTris-buffered saline). The compositions are administered at appropriatedoses, dosing frequency, dosing schedule and route of administration asoptimized for the particular model. Efficacy readouts includemeasurement of FIX activity, PT, aPTT, bleeding time, whole bloodclotting time (WBCT), thrombelastography (TEG or ROTEM), among others.

In one example of a PD model, FIX-XTEN and FIX are administered togenetically-deficient or experimentally-induced HemA or HemB mice. Atvarious time points post-administration, levels of FIX and FIX-XTEN aremeasured by ELISA, activity of FIX and FIX-XTEN are measured bycommercially available FIX activity kit and clotting time is measured byaPTT assay. Overall the results can indicate that the FIX-XTENconstructs may be more efficacious at inhibiting bleeding as compared toFIX and/or equivalent in potency to comparable dosage of FIX with lessfrequent or more convenient dosing intervals.

In a mouse bleeding challenge PD model FIX-XTEN and FIX are administeredto genetically deficient or experimentally induced HemA or Hem B miceand effect on hemostatic challenge is measured. Hemostatic challenge caninclude tail transaction challenge, hemarthropthy challenge, jointbleeding or saphenous vein challenge among others. At various timepoints post administration levels of FIX and FIX-XTEN are measured byELISA, activity of FIX and FIX-XTEN are measured by commerciallyavailable FIX activity kit, bleeding time is measured and clotting timeis measured by aPTT assay. Overall the results can indicate that theFIX-XTEN constructs may be more efficacious at inhibiting bleeding ascompared to FIX and/or equivalent in potency to comparable dosage of FIXwith less frequent or more convenient dosing intervals.

In a dog PD model, FIX-XTEN and FIX are administered togenetically-deficient hemophiliac dogs. At various time pointspost-administration, levels of FIX and FIX-XTEN are measured by ELISA,activity of FIX and FIX-XTEN are measured by commercially available FIXactivity kit and clotting time is measured by aPTT assay. Overall, theresults can indicate that the FIX-XTEN constructs may be moreefficacious at inhibiting bleeding as compared to FIX and/or equivalentin potency to comparable dosage of FIX with less frequent or moreconvenient dosing intervals.

In a dog bleeding challenge PD model FIX a-XTEN and FIX are administeredto genetically-deficient hemophiliac dogs and effect on hemostaticchallenge is measured. Hemostatic challenge can include cuticle bleedingtime, amongst other assays. At various time points post-administrationlevels of FIX and FIX-XTEN are measured by ELISA, activity of FIX andFIX-XTEN are measured by commercially available FIX activity kit,bleeding time is measured and clotting time is measured by aPTT assay.Overall, the results can indicate that the FIX-XTEN constructs may bemore efficacious at inhibiting bleeding as compared to FIX and/orequivalent in potency to comparable dosage of FIX with less frequent ormore convenient dosing intervals.

Additional preclinical models of bleeding include, but are not limitedto, those of hemophilia, surgery, trauma, thrombocytopenia/plateletdysfunction, clopidogrel/heparin-induced bleeding and hydrodynamicinjection. These models can be developed in multiple species, includingmice, rat, rabbits, and dogs using methods equivalent to those used andpublished for other FIX approaches. Overall the results can indicatethat the FIX-XTEN constructs may be more efficacious at inhibitingbleeding as compared to FIX and/or equivalent in potency to comparabledosage of FIX with less frequent or more convenient dosing intervals.

Example 34: CFXTEN with Cleavage Sequences

C-Terminal XTEN Releasable by FXIa

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site cleavage sequence can be incorporated into the FIX-XTENthat contains an amino acid sequence that is recognized and cleaved bythe FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically the aminoacid sequence KLTRAET (SEQ ID NO: 6) is cut after the arginine of thesequence by FXIa protease. FXI is the pro-coagulant protease locatedimmediately before FIX in the intrinsic or contact activated coagulationpathway. Active FXIa is produced from FXI by proteolytic cleavage of thezymogen by FXIIa. Once activated, its natural role in coagulation is toactivate FIX by excising a peptide from zymogen by cutting the proteinat positions R191 and R226 of FIX, which then perpetuates thecoagulation pathway. Production of FXIa is tightly controlled and onlyoccurs when coagulation is necessary for proper hemostasis. Therefore,by incorporation of the KLTRAET cleavage sequence (SEQ ID NO: 6), theXTEN domain would only be removed from FIX concurrent with activation ofthe intrinsic coagulation pathway and when coagulation is requiredphysiologically. This creates a situation where the FIX-XTEN fusionprotein is processed in one additional manner during the activation ofthe intrinsic pathway. In addition to the natural cleavages that occurat R191 and R226 of the FIX domain by FXIa, a third cleavage would occurat the XTEN release site which would decouple the now activated FIXafrom the XTEN protein. In a desirable feature of the inventivecomposition, this creates a situation where FIX-XTEN would remain intactas a pro-drug until activation of coagulation, at which time themolecule is processed to produce free FIXa which reconstitutes oraugments clotting function in a subject in need thereof.

C-Terminal XTEN Releasable by FXIIa

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, theXTEN release site sequence can contain an amino acid sequence that isrecognized and cleaved by the FXIIa protease (EC 3.4.21.38, UniprotP00748). Specifically the sequence TMTRIVGG (SEQ ID NO: 89) is cut afterthe arginine at position 4 of the sequence. FXII is a pro-coagulantprotease located before FIX in the intrinsic or contact activatedcoagulation pathway. Active FXIIa is produced from FXII by contact withnon-self surfaces and by cleavage by kallikrein. Once activated itsnatural role in coagulation is to activate FXI (FIG. 3) by proteolyticcleavage of the zymogen, which then in turn, perpetuates the coagulationpathway. Production of FXIIa is tightly controlled and only occurs whencoagulation is necessary for proper hemostasis. Therefore, byincorporation of the TMTRIVGG cleavage sequence (SEQ ID NO: 89), theXTEN domain would only be removed from FIX concurrent with activation ofthe intrinsic coagulation pathway and when coagulation is requiredphysiologically. This creates a situation where FIX-XTEN fusion isprocessed in one additional manner during the activation of theintrinsic pathway. In addition to the natural cleavages that occur atR191 and R226 of the FIX domain by FXIa, a third cleavage would occur atthe XTEN release site that would decouple the now activated FIXa fromthe XTEN protein. In a desirable feature of the inventive composition,this creates a situation where FIX-XTEN would remain intact as apro-drug until activation of coagulation, at which time the molecule isprocessed to produce free FIXa which reconstitutes or augments clottingfunction in a subject in need thereof.

C-Terminal XTEN Releasable by Kallikrein

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, theXTEN release site sequence can an amino acid sequence that is recognizedand cleaved by the kallikrein protease (EC 3.4.21.34, Uniprot P03952).Specifically the sequence SPFRVVGG (SEQ ID NO: 620) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after the arginine atposition 4 of the sequence. Kallikrein is a pro-coagulant proteaselocated before FIX in the intrinsic or contact activated coagulationpathway. Active Kallikrein is produced from Plasma Kallirien by contactwith non-self surfaces. Once activated its natural role in coagulationis to activate FXII (FIG. 3) by proteolytic cleavage of the zymogen,which then in turn, perpetuates the coagulation pathway. Production ofkallikrien is tightly controlled and only occurs when coagulation isnecessary for proper hemostasis. Therefore, by incorporation of theSPFRVVGG cleavage sequence (SEQ ID NO: 620) the XTEN domain would onlybe removed from FIX concurrent with activation of the intrinsiccoagulation pathway and when coagulation is required physiologically.This creates a situation where FIX-XTEN fusion is processed in oneadditional manner during the activation of the intrinsic pathway. Inaddition to the natural cleavages that occur at R191 and R226 of the FIXdomain by FXIa, a third cleavage would occur at the XTEN release sitethat would decouple the now activated FIXa from the XTEN protein. In adesirable feature of the inventive composition, this creates a situationwhere FIX-XTEN would remain intact as a pro-drug until activation ofcoagulation, at which time the molecule is processed to produce freeFIXa which reconstitutes or augments clotting function in a subject inneed thereof.

C-Terminal XTEN Releasable by FVIIa

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site sequence contains an amino acid sequence that is recognizedand cleaved by the FVIIa protease (EC 3.4.21.21, Uniprot P08709).Specifically the sequence LQVRIVGG (SEQ ID NO: 91) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after the arginine atposition 4 in the sequence. FVIIa is a pro-coagulant protease locatedbefore FIX in the extrinsic or cellular injury activated coagulationpathway. Active FVIIa is produced from FVII in an autocatalytic processaided by binding to tissue factor, phospholipids and calcium. Onceactivated its natural role in coagulation is to activate FIX and FX(FIG. 3) by proteolytic cleavage of the zymogens, which then in turn,perpetuate the coagulation pathway. FVIIa activity is tightly controlledand only occurs when coagulation is necessary for proper hemostasis.Therefore, by incorporation of the LQVRIVGG cleavage sequence (SEQ IDNO: 91) the XTEN domain would only be removed from FIX concurrent withactivation of the intrinsic coagulation pathway and when coagulation isrequired physiologically. This creates a situation where FIX-XTEN fusionis processed in one additional manner during the activation of theintrinsic pathway. In addition to the natural cleavages that would occurat R191 and R226 of the FIX domain by FVIIa, a third cleavage wouldoccur at the XTEN release site which would decouple the now activatedFIXa from the XTEN protein. In a desirable feature of the inventivecomposition, this creates a situation where FIX-XTEN would remain intactas a pro-drug until activation of coagulation, at which time themolecule is processed to produce free FIXa which reconstitutes oraugments clotting function in a subject in need thereof.

C-Terminal XTEN Releasable by FIXa

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site cleavage sequence contains an amino acid sequence that isrecognized and cleaved by the FIXa protease (EC 3.4.21.22, UniprotP00740). Specifically the sequence PLGRIVGG (SEQ ID NO: 92) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after thearginine at position 4 of the sequence. Active FIXa is produced bycleavage of FIX by either FXIa or FVIIa in the presence of phospholipidsand calcium. Once activated its natural role in coagulation is toactivate FX (FIG. 3) by proteolytic cleavage of the zymogen, which thenin turn, perpetuates the coagulation pathway. FIXa activity is tightlycontrolled and only occurs when coagulation is necessary for properhemostasis. Therefore, by incorporation of the PLGRIVGG sequence (SEQ IDNO: 92), the XTEN domain would only be removed from FIX concurrent withactivation of either the extrinsic or intrinsic coagulation pathways,and when coagulation is required physiologically. This creates asituation where FIX-XTEN fusion is processed in one additional mannerduring the activation of the intrinsic pathway. In addition to thenatural cleavages that would occur at R191 and R226 of the FIX domain byFVIIa or FXIa, a third cleavage would occur at the XTEN release sitewhich would decouple the now activated FIXa from the XTEN protein. In adesirable feature of the inventive composition, this creates a situationwhere FIX-XTEN would remain intact as a pro-drug until activation ofcoagulation, at which time the molecule is processed to produce freeFIXa which reconstitutes or augments clotting function in a subject inneed thereof.

C-Terminal XTEN Releasable by FXa

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the FXa protease (EC 3.4.21.6, Uniprot P00742). Specificallythe sequence IEGRTVGG (SEQ ID NO: 93) [Rawlings N. D., et al. (2008)Nucleic Acids Res., 36: D320], is cut after the arginine at position 4in the sequence. Active FXa is produced by cleavage of FX by FIXa in thepresence of phospholipids and calcium and is the step immediately downstream from factor IX in the coagulation pathway. Once activated itsnatural role in coagulation is to activate FII (FIG. 3) by proteolyticcleavage of the zymogen, which then in turn, perpetuates the coagulationpathway. FXa activity is tightly controlled and only occurs whencoagulation is necessary for proper hemostasis. Therefore, byincorporation of the IEGRTVGG sequence (SEQ ID NO: 93), the XTEN domainwould only be removed from FIX concurrent with activation of either theextrinsic or intrinsic coagulation pathways, and when coagulation isrequired physiologically. This creates a situation where FIX-XTEN fusionis processed in one additional manner during the activation of clotting.In addition to the natural cleavages that would occur at R191 and R226of the FIX domain by FVIIa or FXIa, a third cleavage would occur at theXTEN release site which would decouple the now activated FIXa from theXTEN protein. In a desirable feature of the inventive composition, thiscreates a situation where FIX-XTEN would remain intact as a pro-druguntil activation of coagulation, at which time the molecule is processedto produce free FIXa which reconstitutes or augments clotting functionin a subject in need thereof.

C-Terminal XTEN Releasable by FIIa (Thrombin)

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). Specificallythe sequence LTPRSLLV (SEQ ID NO: 94) [Rawlings N. D., et al. (2008)Nucleic Acids Res., 36: D320], is cut after the arginine at position 4in the sequence. Active FIIa is produced by cleavage of FII by FXa inthe presence of phospholipids and calcium and is down stream from factorIX in the coagulation pathway. Once activated its natural role incoagulation is to cleave fibrinogin (FIG. 3), which then in turn, beginsclot formation. FIIa activity is tightly controlled and only occurs whencoagulation is necessary for proper hemostasis. Therefore, byincorporation of the LTPRSLLV sequence (SEQ ID NO: 94), the XTEN domainwould only be removed from FIX concurrent with activation of either theextrinsic or intrinsic coagulation pathways, and when coagulation isrequired physiologically. This creates a situation where FIX-XTEN fusionis processed in one additional manner during the activation ofcoagulation. In addition to the natural cleavages that would occur atR191 and R226 of the FIX domain by FVIIa or FXIa, a third cleavage wouldoccur at the XTEN release site which would decouple the now activatedFIXa from the XTEN protein. In a desirable feature of the inventivecomposition, this creates a situation where FIX-XTEN would remain intactas a pro-drug until activation of coagulation, at which time themolecule is processed to produce free FIXa which reconstitutes oraugments clotting function in a subject in need thereof.

C-Terminal XTEN Releasable by Elastase-2

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the elastase-2 protease (EC 3.4.21.37, Uniprot P08246).Specifically the sequence LGPVSGVP (SEQ ID NO: 95) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in thesequence. Elastase is constitutively expressed by neutrophils and ispresent at all times in the circulation. Its activity is tightlycontrolled by serpins and is therefore minimally active most of thetime. Therefore as the long lived FIX-XTEN circulates, a fraction of itis cleaved, creating a pool of shorter-lived FIX to be used incoagulation. In a desirable feature of the inventive composition, thiscreates a circulating pro-drug depot that constantly releases aprophylactic amount of FIX.

C-Terminal XTEN Releasable by MMP-12

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900).Specifically the sequence GPAGLGGA (SEQ ID NO: 97) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 of thesequence. MMP-12 is constitutively expressed in whole blood. Thereforeas the long lived FIX-XTEN circulates, a fraction of it is cleaved,creating a pool of shorter-lived FIX to be used in coagulation. In adesirable feature of the inventive composition, this creates acirculating pro-drug depot that constantly releases a prophylacticamount of FIX.

C-Terminal XTEN Releasable by MMP-13

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the MMP-13 protease (EC 3.4.24.-, Uniprot P45452).Specifically the sequence GPAGLRGA (SEQ ID NO: 99) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after position 4.MMP-13 is constitutively expressed in whole blood. Therefore as the longlived FIX-XTEN circulates, a fraction of it is cleaved, creating a poolof shorter-lived FIX to be used in coagulation. In a desirable featureof the inventive composition, this creates a circulating pro-drug depotthat constantly releases a prophylactic amount of FIX.

C-Terminal XTEN Releasable by MMP-17

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot Q9ULZ9).Specifically the sequence APLGLRLR (SEQ ID NO: 101) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in thesequence. MMP-17 is constitutively expressed in whole blood. Thereforeas the long lived FIX-XTEN circulates, a fraction of it is cleaved,creating a pool of shorter-lived FIX to be used in coagulation. In adesirable feature of the inventive composition, this creates acirculating pro-drug depot that constantly releases a prophylacticamount of FIX.

C-Terminal XTEN Releasable by MMP-20

An FIX-XTEN fusion protein consisting of an XTEN protein fused to theC-terminus of FIX can be created with a XTEN release site cleavagesequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, therelease site contains an amino acid sequence that is recognized andcleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot 060882).Specifically the sequence PALPLVAQ (SEQ ID NO: 102) [Rawlings N. D., etal. (2008) Nucleic Acids Res., 36: D320], is cut after position 4(depicted by the arrow). MMP-20 is constitutively expressed in wholeblood. Therefore as the long lived FIX-XTEN circulates, a fraction of itis cleaved, creating a pool of shorter-lived FIX to be used incoagulation. In a desirable feature of the inventive composition, thiscreates a circulating pro-drug depot that constantly releases aprophylactic amount of FIX.

Optimization of the Release Rate of C-Terminal XTEN

Variants of the foregoing Examples can be created in which the releaserate of C-terminal XTEN 15 altered. As the rate of XTEN release by anXTEN release protease is dependent on the sequence of the XTEN releasesite, by varying the amino acid sequence in the XTEN release site onecan control the rate of XTEN release. The sequence specificity of manyproteases is well known in the art, and is documented in several databases. In this case, the amino acid specificity of proteases is mappedusing combinatorial libraries of substrates [Harris, J. L., et al.(2000) Proc Natl Acad Sci USA, 97: 7754] or by following the cleavage ofsubstrate mixtures as illustrated in [Schellenberger, V., et al. (1993)Biochemistry, 32: 4344]. An alternative is the identification of optimalprotease cleavage sequences by phage display [Matthews, D., et al.(1993) Science, 260: 1113]. Constructs is made with variant sequencesand assayed for XTEN release using standard assays for detection of theXTEN polypeptides.

Example 35: Integration of XTEN Internal to the CF Sequence

Internal XTEN Fusion into the KNSADK Loop (SEQ ID NO: 621)

An FIX-XTEN fusion protein consisting of an XTEN protein inserted into aloop of FIX can be created, as depicted in FIG. 2F. Specifically, theXTEN sequence is inserted as a fusion into the KNSADNK loop (SEQ ID NO:622) of the EGF2 domain (residues 146-152), which has no knownhemophilia B mutations and is not highly structured in the FIX crystalstructure. In this case, the insertion is made by dividing the nativesequence at the SA bond of the loop sequence and fusing the XTENsequence into the gap. This would give rise to a loop sequenceKNS-XTEN-ADNK (‘ADNK’ disclosed as SEQ ID NO: 623). In a desirablefeature of the inventive composition, this creates a situation whereFIX-XTEN would remain intact as a pro-drug until activation ofcoagulation, at which time the molecule is processed to produceFIXa-XTEN, which reconstitutes or augments clotting function in asubject in need thereof.

Internal XTEN Fusion into the LAEN Loop (SEQ ID NO: 624)

An FIX-XTEN fusion protein consisting of an XTEN protein inserted into aloop of FIX can be created, as depicted in FIG. 2F. Specifically, theXTEN sequence is inserted as a fusion into the LAEN loop (SEQ ID NO:624) of the EGF2 domain (residues 163-166), which has no knownhemophilia B mutations and is not highly structured in the FIX crystalstructure. In this case, the insertion is made by dividing the nativesequence at the AE bond of the sequence and fusing the XTEN sequenceinto the gap. This would give rise to a loop sequence LA-XTEN-EN. In adesirable feature of the inventive composition, this creates a situationwhere FIX-XTEN would remain intact as a pro-drug until activation ofcoagulation, at which time the molecule is processed to produceFIXa-XTEN, which reconstitutes or augments clotting function in asubject in need thereof.

Internal XTEN Fusion into the Activation Peptide

An FIX-XTEN fusion protein consisting of an XTEN protein inserted into aloop of FIX can be created, as depicted in FIG. 2D. Specifically, theXTEN fusion is placed into the activation peptide (residues 192-226)such that neither of the two native FXIa cleavage sites is disrupted.The insertion is made by dividing the native sequence at the T209-I210bond of the sequence and fusing the XTEN sequence into the gap. Thisgives rise to a sequence starting at residue 188 ofKLTRAETVFPDVDYVNSTEAET-XTEN-ILDNITQSTQSFNDFTRVVGGE (SEQ ID NOS 625 and626, respectively). FXI is the pro-coagulant protease locatedimmediately before FIX in the intrinsic or contact activated coagulationpathway. Active FXIa is produced from FXI by proteolytic cleavage of thezymogen by FXIIa. Once activated its natural role in coagulation is toactivate FIX (FIG. 8) by excising the activation peptide from the FIXzymogen by cutting the protein at positions R191 and R226. These cutssites are depicted by arrows and the sequences are designed to leave theP4-P4′ sites unaltered to allow for natural cleavage activity during thecoagulation cascade. Therefore the XTEN domain would only be removedfrom FIX as part of the normal activation process within the intrinsiccoagulation pathway.

Internal XTEN Fusion in Between the FIX EGF Domains

An FIX-XTEN fusion protein consisting of an XTEN protein inserted into aloop of FIX can be created, as depicted in FIG. 2C. Specifically, theXTEN fusion is placed in between the two EGF like domains of FIX(junction is between residues 129 and 130). The insertion is made bydividing the native sequence at the E129-L130 bond and fusing the XTENsequence into the gap. This would give rise to a sequence starting atresidue 121 of FGFEGKNCE-XTEN-LDVTCNIKNGR (SEQ ID NOS 627 and 628,respectively). Practically, this creates a situation where FIX-XTENwould circulate intact until activation of coagulation, at which timethe molecule is processed to produce FIXa-XTEN, which reconstitutes oraugments clotting function in an individual.

Example 36: Human Clinical Trial Designs for Evaluating CFXTENComprising FVIIa

NovoSeven® is recombinant human coagulation factor VIIa (rFVIIa),intended for promoting hemostasis by activating the extrinsic pathway ofthe coagulation cascade. Due to its short half-life, NovoSeven is dosedintravenously from every 2 to every 6 hours until hemostasis isachieved. Fusion of XTEN to FVII improves the half-life of the protein,thus enabling a reduced dosing frequency using such FVII-containingfusion protein compositions.

Clinical trials can be designed such that the efficacy and advantages ofFVIIa-XTEN, relative to NovoSeven, can be verified in humans. Forexample, the FVIIa-XTEN, can be used in clinical trials for treatment ofbleeding as performed for NovoSeven. Such studies would comprise threephases. First, a Phase I safety and pharmacokinetics study in adultpatients is conducted to determine the maximum tolerated dose andpharmacokinetics and pharmacodynamics in humans (either normal subjectsor patients with hemophilia), as well as to define potential toxicitiesand adverse events to be tracked in future studies. The study isconducted in which single rising doses of FVIIa-XTEN compositions isadministered and biochemical, PK, and clinical parameters is measured.This would permit the determination of the maximum tolerated dose andestablish the threshold and maximum concentrations in dosage andcirculating drug that constitute the therapeutic window for therespective components. Thereafter, clinical trials is conducted inpatients with the disease, disorder or condition.

Clinical trials could be conducted in patients suffering from anydisease in which NovoSeven may be expected to provide clinical benefit.For example, such indications include bleeding episodes in hemophilia Aor B, patients with inhibitors to factor VIII or factor IX, and inpatients with acquired hemophilia, prevention of bleeding in surgicalinterventions or invasive procedures in hemophilia A or B patients withinhibitors to factor VIII or factor IX and in patients with acquiredhemophilia, treatment of bleeding episodes in patients with congenitalFVII deficiency, and prevention of bleeding in surgical interventions orinvasive procedures in patients with congenital FVII deficiency.FVIIa-XTEN may also be indicated for use in additional patientpopulations. Parameters and clinical endpoints are measured as afunction of the dosing of the fusion proteins compositions, yieldingdose-ranging information on doses that is appropriate for a subsequentPhase III trial, in addition to collecting safety data related toadverse events. The PK parameters are correlated to the physiologic,clinical and safety parameter data to establish the therapeutic windowand the therapeutic dose regimen for the FVII-XTEN composition,permitting the clinician to establish the appropriate dose ranges forthe composition. Finally, a phase III efficacy study is conductedwherein patients is administered the FVII-XTEN composition at the doseregimen, and a positive control (such as a commercially-availableNovoSeven), or a placebo is administered using a dosing schedule deemedappropriate given the pharmacokinetic and pharmacodynamic properties ofthe respective compositions, with all agents administered for anappropriately extended period of time to achieve the study endpoints.Parameters that are monitored include PT assay, bleeding time assay,control of bleeding episodes, or the occurrence of spontaneous bleedingepisodes; parameters that is tracked relative to the placebo or positivecontrol groups. Efficacy outcomes is determined using standardstatistical methods. Toxicity and adverse event markers are also befollowed in this study to verify that the compound is safe when used inthe manner described.

Example 37: Human Clinical Trial Designs for Evaluating CFXTENComprising FIX

BeneFIX®, Coagulation Factor IX (Recombinant), is indicated for thecontrol and prevention of hemorrhagic episodes in patients withhemophilia B (congenital factor IX deficiency or Christmas disease),including control and prevention of bleeding in surgical settings.Dosage and duration of treatment for all factor IX products depend onthe severity of the factor IX deficiency, the location and extent ofbleeding, and the patient's clinical condition, age and recovery offactor IX. Fusion of XTEN to FIX improves the half-life of the protein,thus enabling a reduced dosing frequency.

Clinical trials can be designed such that the efficacy and advantages ofFIX-XTEN, relative to other factor IX clinical products, can be verifiedin humans. For example, the FIX-XTEN, can be used in clinical trials fortreatment of hemorrhagic episodes as performed for Benefix. Such studieswould comprise three phases. First, a Phase I safety andpharmacokinetics study in adult patients is conducted to determine themaximum tolerated dose and pharmacokinetics and pharmacodynamics inhumans (either normal subjects or patients with hemophilia), as well asto define potential toxicities and adverse events to be tracked infuture studies. The study is conducted in which single rising doses ofFIX-XTEN compositions is administered and biochemical, PK, and clinicalparameters is measured. This would permit the determination of themaximum tolerated dose and establish the threshold and maximumconcentrations in dosage and circulating drug that constitute thetherapeutic window for the respective components. Thereafter, clinicaltrials is conducted in patients with the disease, disorder or condition.

Clinical trials could be conducted in patients suffering from anydisease in which factor IX may be expected to provide clinical benefit.For example, such indications include the control and prevention ofhemorrhagic episodes in patients with hemophilia B (congenital factor IXdeficiency or Christmas disease), including control and prevention ofbleeding in surgical settings. FIX-XTEN may also be indicated for use inadditional patient populations. Parameters and clinical endpoints aremeasured as a function of the dosing of the fusion proteinscompositions, yielding dose-ranging information on doses that isappropriate for a subsequent Phase III trial, in addition to collectingsafety data related to adverse events. The PK parameters are correlatedto the physiologic, clinical and safety parameter data to establish thetherapeutic window and the therapeutic dose regimen for the FIX-XTENcomposition, permitting the clinician to establish the appropriate doseranges for the composition. Finally, a phase III efficacy study isconducted wherein patients is administered the FIX-XTEN composition atthe dose regimen, and a positive control (such as acommercially-available BeneFIX), or a placebo is administered using adosing schedule deemed appropriate given the pharmacokinetic andpharmacodynamic properties of the respective compositions, with allagents administered for an appropriately extended period of time toachieve the study endpoints. Parameters that are monitored include aPTTassay, bleeding time assay, control of bleeding episodes, or theoccurrence of spontaneous bleeding episodes; parameters that is trackedrelative to the placebo or positive control groups. Efficacy outcomes isdetermined using standard statistical methods. Toxicity and adverseevent markers are also be followed in this study to verify that thecompound is safe when used in the manner described.

Example 38: Analytical Size Exclusion Chromatography of XTEN FusionProteins with Diverse Payloads

Size exclusion chromatography analyses were performed on fusion proteinscontaining various therapeutic proteins and unstructured recombinantproteins of increasing length. An exemplary assay used a TSKGel-G4000SWXL (7.8 mm×30 cm) column in which 40 μg of purified glucagon fusionprotein at a concentration of 1 mg/ml was separated at a flow rate of0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profileswere monitored using OD214 nm and OD280 nm. Column calibration for allassays were performed using a size exclusion calibration standard fromBioRad; the markers include thyroglobulin (670 kDa), bovinegamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin(17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographicprofiles of Glucagon-Y288, Glucagon-Y144, Glucagon-Y72, Glucagon-Y36 areshown as an overlay in FIG. 35. The data show that the apparentmolecular weight of each compound is proportional to the length of theattached XTEN sequence. However, the data also show that the apparentmolecular weight of each construct is significantly larger than thatexpected for a globular protein (as shown by comparison to the standardproteins run in the same assay). Based on the SEC analyses for allconstructs evaluated, including a CFXTEN composition, the apparentmolecular weights, the apparent molecular weight factor (expressed asthe ratio of apparent molecular weight to the calculated molecularweight) and the hydrodynamic radius (RH in nm) are shown in Table 31.The results indicate that incorporation of different XTENs of 576 aminoacids or greater confers an apparent molecular weight for the fusionprotein of approximately 339 kDa to 760, and that XTEN of 864 aminoacids or greater confers an apparent molecular weight greater thanapproximately 800 kDA. The results of proportional increases in apparentmolecular weight to actual molecular weight were consistent for fusionproteins created with XTEN from several different motif families; i.e.,AD, AE, AF, AG, and AM, with increases of at least four-fold and ratiosas high as about 17-fold. Additionally, the incorporation of XTEN fusionpartners with 576 amino acids or more into fusion proteins with thevarious payloads (and 288 residues in the case of glucagon fused toY288) resulted with a hydrodynamic radius of 7 nm or greater; wellbeyond the glomerular pore size of approximately 3-5 nm. Accordingly, itis expected that fusion proteins comprising growth and XTEN have reducedrenal clearance, contributing to increased terminal half-life andimproving the therapeutic or biologic effect relative to a correspondingun-fused biologic payload protein.

TABLE 31 SEC analysis of various polypeptides XTEN or Apparent Constructfusion Therapeutic Actual Apparent Molecular Name partner Protein MW(kDa) MW (kDa) Weight Factor Rh (nm) AC14 Y288 Glucagon 28.7 370 12.97.0 AC28 Y144 Glucagon 16.1 117 7.3 5.0 AC34 Y72 Glucagon 9.9 58.6 5.93.8 AC33 Y36 Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 Glucagon 14.1 76.4 5.44.3 AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.25.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1 7.7AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6 AC398AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7 10.1 AC85AE864 Exendin-4 83.6 938 11.2 8.9 AC114 AM875 Exendin-4 82.4 1344 16.39.4 AC143 AM875 CF 100.6 846 8.4 8.7 AC227 AM875 IL-lra 95.4 1103 11.69.2 AC228 AM1318 IL-lra 134.8 2286 17.0 10.5

Example 39: Pharmacokinetics of Extended Polypeptides Fused to GFP inCynomolgus Monkeys

The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-XTENY576 and XTEN_AD836-GFP were tested in cynomolgus monkeys to determinethe effect of composition and length of the unstructured polypeptides onPK parameters. Blood samples were analyzed at various times afterinjection and the concentration of GFP in plasma was measured by ELISAusing a polyclonal antibody against GFP for capture and a biotinylatedpreparation of the same polyclonal antibody for detection. Results aresummarized in FIG. 25. They show a surprising increase of half-life withincreasing length of the XTEN sequence. For example, a half-life of 10 hwas determined for GFP-XTEN_L288 (with 288 amino acid residues in theXTEN). Doubling the length of the unstructured polypeptide fusionpartner to 576 amino acids increased the half-life to 20-22 h formultiple fusion protein constructs; i.e., GFP-XTEN_L576, GFP-XTEN AF576,GFP-XTEN Y576. A further increase of the unstructured polypeptide fusionpartner length to 836 residues resulted in a half-life of 72-75 h forXTEN_AD836-GFP. Thus, increasing the polymer length by 288 residues from288 to 576 residues increased in vivo half-life by about 10 h. However,increasing the polypeptide length by 260 residues from 576 residues to836 residues increased half-life by more than 50 h. These results showthat there is a surprising threshold of unstructured polypeptide lengththat results in a greater than proportional gain in in vivo half-life.Thus, fusion proteins comprising extended, unstructured polypeptides areexpected to have the property of enhanced pharmacokinetics compared topolypeptides of shorter lengths.

Example 40: Serum Stability of XTEN

A fusion protein containing XTEN_AE864 fused to the N-terminus of GFPwas incubated in monkey plasma and rat kidney lysate for up to 7 days at37° C. Samples were withdrawn at time 0, Day 1 and Day 7 and analyzed bySDS PAGE followed by detection using Western analysis and detection withantibodies against GFP as shown in FIG. 26. The sequence of XTEN_AE864showed negligible signs of degradation over 7 days in plasma. However,XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days. The invivo stability of the fusion protein was tested in plasma sampleswherein the GFP_AE864 was immunoprecipitated and analyzed by SDS PAGE asdescribed above. Samples that were withdrawn up to 7 days afterinjection showed very few signs of degradation. The results demonstratethe resistance of CFXTEN to degradation due to serum proteases; a factorin the enhancement of pharmacokinetic properties of the CFXTEN fusionproteins.

Example 41: Increasing Solubility and Stability of a Peptide Payload byLinking to XTEN

In order to evaluate the ability of XTEN to enhance thephysical/chemical properties of solubility and stability, fusionproteins of glucagon plus shorter-length XTEN were prepared andevaluated. The test articles were prepared in Tris-buffered saline atneutral pH and characterization of the Gcg-XTEN solution was byreverse-phase HPLC and size exclusion chromatography to affirm that theprotein was homogeneous and non-aggregated in solution. The data arepresented in Table 32. For comparative purposes, the solubility limit ofunmodified glucagon in the same buffer was measured at 60 μM (0.2mg/mL), and the result demonstrate that for all lengths of XTEN added, asubstantial increase in solubility was attained. Importantly, in mostcases the glucagon-XTEN fusion proteins were prepared to achieve targetconcentrations and were not evaluated to determine the maximumsolubility limits for the given construct. However, in the case ofglucagon linked to the AF-144 XTEN, the limit of solubility wasdetermined, with the result that a 60-fold increase in solubility wasachieved, compared to glucagon not linked to XTEN. In addition, theglucagon-AF144 CFXTEN was evaluated for stability, and was found to bestable in liquid formulation for at least 6 months under refrigeratedconditions and for approximately one month at 37° C. (data not shown).

The data support the conclusion that the linking of short-length XTENpolypeptides to a biologically active protein such as glucagon canmarkedly enhance the solubility properties of the protein by theresulting fusion protein, as well as confer stability at the higherprotein concentrations.

TABLE 32 Solubility of Glucagon-XTEN constructs Test Article SolubilityGlucagon 60 μM Glucagon-Y36 >370 μM Glucagon-Y72 >293 μMGlucagon-AF108 >145 μM Glucagon-AF120 >160 μM Glucagon-Y144 >497 μMGlucagon-AE144 >467 μM Glucagon-AF144 >3600 μM Glucagon-Y288 >163 μM

Example 42: Analysis of Sequences for Secondary Structure by PredictionAlgorithms

Amino acid sequences can be assessed for secondary structure via certaincomputer programs or algorithms, such as the well-known Chou-Fasmanalgorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and theGarnier-Osguthorpe-Robson, or “GOR” method (Gamier J, Gibrat J F, RobsonB. (1996). GOR method for predicting protein secondary structure fromamino acid sequence. Methods Enzymol 266:540-553). For a given sequence,the algorithms can predict whether there exists some or no secondarystructure at all, expressed as total and/or percentage of residues ofthe sequence that form, for example, alpha-helices or beta-sheets or thepercentage of residues of the sequence predicted to result in randomcoil formation.

Several representative sequences from XTEN “families” have been assessedusing two algorithm tools for the Chou-Fasman and GOR methods to assessthe degree of secondary structure in these sequences. The Chou-Fasmantool was provided by William R. Pearson and the University of Virginia,at the “Biosupport” internet site, URL located on the World Wide Web atfasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it existedon Jun. 19, 2009. The GOR tool was provided by Pole InformatiqueLyonnais at the Network Protein Sequence Analysis internet site, URLlocated on the World Wide Web at.npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl as it existed on Jun. 19,2008.

As a first step in the analyses, a single XTEN sequence was analyzed bythe two algorithms. The AE864 composition is a XTEN with 864 amino acidresidues created from multiple copies of four 12 amino acid sequencemotifs consisting of the amino acids G, S, T, E, P, and A. The sequencemotifs are characterized by the fact that there is limitedrepetitiveness within the motifs and within the overall sequence in thatthe sequence of any two consecutive amino acids is not repeated morethan twice in any one 12 amino acid motif, and that no three contiguousamino acids of full-length the XTEN are identical. Successively longerportions of the AF 864 sequence from the N-terminus were analyzed by theChou-Fasman and GOR algorithms (the latter requires a minimum length of17 amino acids). The sequences were analyzed by entering the FASTAformat sequences into the prediction tools and running the analysis. Theresults from the analyses are presented in Table 33.

The results indicate that, by the Chou-Fasman calculations, the fourmotifs of the AE family (Table 1) have no alpha-helices or beta sheets.The sequence up to 288 residues was similarly found to have noalpha-helices or beta sheets. The 432 residue sequence is predicted tohave a small amount of secondary structure, with only 2 amino acidscontributing to an alpha-helix for an overall percentage of 0.5%. Thefull-length AF864 polypeptide has the same two amino acids contributingto an alpha-helix, for an overall percentage of 0.2%. Calculations forrandom coil formation revealed that with increasing length, thepercentage of random coil formation increased. The first 24 amino acidsof the sequence had 91% random coil formation, which increased withincreasing length up to the 99.77% value for the full-length sequence.

Numerous XTEN sequences of 500 amino acids or longer from the othermotif families were also analyzed and revealed that the majority hadgreater than 95% random coil formation. The exceptions were thosesequences with one or more instances of three contiguous serineresidues, which resulted in predicted beta-sheet formation. However,even these sequences still had approximately 99% random coil formation.

In contrast, a polypeptide sequence of 84 residues limited to A, S, andP amino acids was assessed by the Chou-Fasman algorithm, which predicteda high degree of predicted alpha-helices. The sequence, which hadmultiple repeat “AA” and “AAA” sequences, had an overall predictedpercentage of alpha-helix structure of 69%. The GOR algorithm predicted78.57% random coil formation; far less than any sequence consisting of12 amino acid sequence motifs consisting of the amino acids G, S, T, E,P, analyzed in the present Example.

The analysis supports the conclusion that: 1) XTEN created from multiplesequence motifs of G, S, T, E, P, and A that have limited repetitivenessas to contiguous amino acids are predicted to have very low amounts ofalpha-helices and beta-sheets; 2) that increasing the length of the XTENdoes not appreciably increase the probability of alpha-helix orbeta-sheet formation; and 3) that progressively increasing the length ofthe XTEN sequence by addition of non-repetitive 12-mers consisting ofthe amino acids G, S, T, E, P, and A results in increased percentage ofrandom coil formation. In contrast, polypeptides created from aminoacids limited to A, S and P that have a higher degree of internalrepetitiveness are predicted to have a high percentage of alpha-helices,as determined by the Chou-Fasman algorithm, as well as random coilformation. Based on the numerous sequences evaluated by these methods,it is concluded that XTEN created from sequence motifs of G, S, T, E, P,and A that have limited repetitiveness (defined as no more than twoidentical contiguous amino acids in any one motif) greater than about400 amino acid residues in length are expected to have very limitedsecondary structure. With the exception of motifs containing threecontiguous serines, it is believed that any order or combination ofsequence motifs from Table 3 can be used to create an XTEN polypeptideof a length greater than about 400 residues that will result in an XTENsequence that is substantially devoid of secondary structure. Suchsequences are expected to have the characteristics described in theCFXTEN embodiments of the invention disclosed herein.

TABLE 33CHOU-FASMAN and GOR prediction calculations of polypeptide sequences SEQSEQ ID No. Chou-Fasman GOR NAME Sequence NO: Residues CalculationCalculation GSTSESPSGTAP 629   12 Residue totals*: H: 0 E: 0 Notpercent: H: 0.0 E: 0.0 Determined GTSTPESGSASP 630   12Residue totals: H: 0 E: 0 Not percent: H: 0.0 E: 0.0 DeterminedGTSPSGESSTAP 631   12 Residue totals: H: 0 E: 0 Notpercent: H: 0.0 E: 0.0 Determined GSTSSTAESPGP 632   12Residue totals: H: 0 E: 0 Not percent: H: 0.0 E: 0.0 DeterminedGSPAGSPTSTEEGTSESATPES 633   24 Residue totals: H: 0 E: 0 91.67% GPpercent: H: 0.0 E: 0.0 GSPAGSPTSTEEGTSESATPES 634   36Residue totals: H: 0 E: 0 94.44% GPGTSTEPSEGSAP percent: H: 0.0 E: 0.0GSPAGSPTSTEEGTSESATPES 635   48 Residue totals: H: 0 E: 0 93.75%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.0 E: 0.0 STEEGSPAGSPTSTEEGTSESATPES 636   60 Residue totals: H: 0 E: 0 96.67%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.0 E: 0.0 STEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES 637  108 Residue totals: H: 0 E: 0 97.22%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.0 E: 0.0 STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSESATPES 638 216 Residue totals: H: 0 E: 0 99.07% GPGTSTEPSEGSAPGSPAGSPTpercent: H: 0.0 E: 0.0 STEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES 639  432 Residue totals: H: 2 E: 3 99.54%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.5 E: 0.7 STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAP AE864 GSPAGSPTSTEEGTSESATPES 640 864 Residue totals: H: 2 E: 3 99.77% GPGTSTEPSEGSAPGSPAGSPTpercent: H: 0.2 E: 0.3 STEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT STEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTSTEP SEGSAP AD576GSSESGSSEGGPGSGGEPSES 641  576 Residue totals: H: 7 E: 0 99.65%GSSGSSESGSSEGGPGSSESGS percent: H: 1.2 E: 0.0 SEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGE SPGGSSGSESGSEGSSGPGESS GSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPS ESGSSGESPGGSSGSESGESPG GSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSS GSGGEPSESGSSGSEGSSGPG ESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGG EPSESGSSGSSESGSSEGGPGE SPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGS ESGESPGGSSGSESGSSESGSS EGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSG GEPSESGSSGSSESGSSEGGPG SGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSE GGPGSGGEPSESGSSGSSESGS SEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGS EGSSGPGESSGSSESGSSEGGP GSEGSSGPGESS AE576GSPAGSPTSTEEGTSESATPES 642  576 Residue totals: H: 2 E: 0 99.65%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.4 E: 0.0 STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGT STEPSEGSAPGTSTEPSEGSAPGPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAP AF540 GSTSSTAESPGPGSTSSTAESP 643  540Residue totals: H: 2 E: 0 99.65 GPGSTSESPSGTAPGSTSSTAEpercent: H: 0.4 E: 0.0 SPGPGSTSSTAESPGPGTSTPE SGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS TSESPSGTAPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPS GTAPGSTSESPSGTAPGSTSES PSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGS TSSTAESPGPGSTSSTAESPGP GTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESG SASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT STPESGSASPGTSTPESGSASP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSTPE SGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGS TSSTAESPGPGTSTPESGSASP GSTSESPSGTAP AF504GASPGTSSTGSPGSSPSASTGT 644  504 Residue totals: H: 0 E: 0 94.44%GPGSSPSASTGTGPGTPGSGT percent: H: 0.0 E: 0.0 ASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTG PGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG PGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT GSPGSSPSASTGTGPGASPGTS STGSP AE864GSPAGSPTSTEEGTSESATPES 645  864 Residue totals: H: 2 E: 3 99.77%GPGTSTEPSEGSAPGSPAGSPT percent: H: 0.2 E: 0.4 STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE SATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAP AF864 GSTSESPSGTAPGTSPSGESST 646  875 Residue totals: H: 2 E: 095.20% APGSTSESPSGTAPGSTSESPS percent: H: 0.2 E: 0.0GTAPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAP GTSPSGESSTAPGSTSSTAESP GPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPE SGSASPGTSTPESGSASPGSTS ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGP GTSTPESGSASPGSTSESPSGT APGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE SGSASPGSTSSTAESPGPGSTS STAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGT APGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSES PSGTAPGSTSESPSGTAPGSTS ESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASP GTSPSGESSTAPGTSPSGESST APGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGTSP SGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASP GSTSESPSGTAPGTSTPESGSA SPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSG ESSTAPGSTSSTAESPGPGTSP SGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAP GTSPSGESSTAPGSTSSTAESP GPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPS GATGSPGSSTPSGATGSP AG864 GGSPGASPGTSSTGSPGSSPSA647  868 Residue totals: H: 0 E: 0 94.70% STGTGPGSSPSASTGTGPGTPpercent: H: 0.0 E: 0.0 GSGTASSSPGSSTPSGATGSPG SNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG ASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSG ATGSPGSSTPSGATGSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS PGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGT SSTGSPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GASPGTSSTGSPGSSTPSGAT GSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPAM875 GTSTEPSEGSAPGSEPATSGSE 648  875 Residue totals: H: 7 E: 3 98.63%TPGSPAGSPTSTEEGSTSSTAE percent: H: 0.8 E: 0.3 SPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTST PESGSASPGTSTPESGSASPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE SGPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGASASG APSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS TSSTAESPGPGSTSESPSGTAP GTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPG TSPSGESSTAPGSEPATSGSET PGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPES GSASPGSTSESPSGTAPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPG SSPSASTGTGPGASPGTSSTGS PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSG ATGSPGSSPSASTGTGPGASP GTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318 GTSTEPSEGSAPGSEPATSGSE 649 1318Residue totals: H: 7 E: 0 99.17% TPGSPAGSPTSTEEGSTSSTAEpercent: H: 0.7 E: 0.0 SPGPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS EPATSGSETPGTSESATPESGP GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSSTP SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSET PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGP APSGGSEPATSGSETPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSEEGS PAGSPTSEEGTSESATPESGP GSPAGSPTSEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPS GTAPGTSPSGESSTAPGSTSES PSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSEPSEGSAPGT SESATPESGPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGTSTEP SEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGT STEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGA TGSPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPG STSSTAESPGPGTSPSGESSTA PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSAS TGTGPGSSTPSGATGSPGASP GTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAP GTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPE SGSASPGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGSSTPSGATG SPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSG ESSTAPGSTSSTAESPGPGSST PSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTE EGSPAGSPTSTEEGTSTEPSEG SAP AM923MAEPAGSPTSTEEGASPGTSS 650  924 Residue totals: H: 4 E: 3 98.70%TGSPGSSTPSGATGSPGSSTPS percent: H: 0.4 E: 0.3 GATGSPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGS TSSTAESPGPGTSTPESGSASP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESG SASPGSEPATSGSETPGTSESA TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGS EPATSGSETPGSPAGSPTSTEE GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPS EGSAPGTSTEPSEGSAPGSEPA TSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG ASASGAPSTGGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESP SGTAPGTSPSGESSTAPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPG TSESATPESGPGSEPATSGSET PGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATS GSETPGSEPATSGSETPGTSTE PSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG TSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGTSESATPES GPGTSTEPSEGSAPGTSTEPSE GSAPAE912 MAEPAGSPTSTEEGTPGSGTA 651  913 Residue totals: H: 8 E: 3 99.45%SSSPGSSTPSGATGSPGASPGT percent: H: 0.9 E: 0.3 SSTGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSPAG SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG TSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT PESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESATPESG PGTSTEPSEGSAP BC864GTSTEPSEPGSAGTSTEPSEPG 652 Residue totals: H: 0 E: 0 99.77%SAGSEPATSGTEPSGSGASEPT percent: H: 0 E: 0 STEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGA SEPTSTEPGTSTEPSEPGSAGS EPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTE PSGTSTEPSEPGSAGTSTEPSE PGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGA SEPTSTEPGTSEPSTSEPGAGS EPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPG SAGSGASEPTSTEPGSEPATSG TEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTST EPSEPGSAGSEPATSGTEPSGS GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTST EPGTSTEPSEPGSAGSGASEPT STEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGA SEPTSTEPGTSTEPSEPGSAGS EPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTE PSGTSTEPSEPGSAGSEPATSG TEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTST EPSEPGSAGTSTEPSEPGSAGT STEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPG SAGTSTEPSEPGSAGTSTEPSE PGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSE PATSGTEPSGSEPATSGTEPSG SEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTS TEPGTSTEPSEPGSAGSEPATS GTEPSGSGASEPTSTEPGTSTEPSEPGSA ASPAAPAPASPAAPAPSAPAA 653   84 Residue totals: H: 58 E: 0 78.57%APASPAPAAPSAPAPAAPSAA percent: H: 69.0 E: 0.0 SPAAPSAPPAAASPAAPSAPPAASAAAPAAASAAASAPSAA A *H: alpha-helix E: beta-sheet

Example 43: Analysis of Polypeptide Sequences for Repetitiveness

Polypeptide amino acid sequences can be assessed for repetitiveness byquantifying the number of times a shorter subsequence appears within theoverall polypeptide. For example, a polypeptide of 200 amino acidresidues has 192 overlapping 9-amino acid subsequences (or 9-mer“frames”), but the number of unique 9-mer subsequences will depend onthe amount of repetitiveness within the sequence. In the presentanalysis, different sequences were assessed for repetitiveness bysumming the occurrence of all unique 3-mer subsequences for each 3-aminoacid frame across the first 200 amino acids of the polymer portiondivided by the absolute number of unique 3-mer subsequences within the200 amino acid sequence. The resulting subsequence score is a reflectionof the degree of repetitiveness within the polypeptide.

The results, shown in Table 34, indicate that the unstructuredpolypeptides consisting of 2 or 3 amino acid types have high subsequencescores, while those of consisting of 12 amino acids motifs of the sixamino acids G, S, T, E, P, and A with a low degree of internalrepetitiveness, have subsequence scores of less than 10, and in somecases, less than 5. For example, the L288 sequence has two amino acidtypes and has short, highly repetitive sequences, resulting in asubsequence score of 50.0. The polypeptide J288 has three amino acidtypes but also has short, repetitive sequences, resulting in asubsequence score of 33.3. Y576 also has three amino acid types, but isnot made of internal repeats, reflected in the subsequence score of 15.7over the first 200 amino acids. W576 consists of four types of aminoacids, but has a higher degree of internal repetitiveness, e.g., “GGSG”(SEQ ID NO: 654), resulting in a subsequence score of 23.4. The AD576consists of four types of 12 amino acid motifs, each consisting of fourtypes of amino acids. Because of the low degree of internalrepetitiveness of the individual motifs, the overall subsequence scoreover the first 200 amino acids is 13.6. In contrast, XTEN's consistingof four motifs contains six types of amino acids, each with a low degreeof internal repetitiveness have lower subsequence scores; i.e., AE864(6.1), AF864 (7.5), and AM875 (4.5).

Conclusions: The results indicate that the combination of 12 amino acidsubsequence motifs, each consisting of four to six amino acid types thatare essentially non-repetitive, into a longer XTEN polypeptide resultsin an overall sequence that is non-repetitive. This is despite the factthat each subsequence motif may be used multiple times across thesequence. In contrast, polymers created from smaller numbers of aminoacid types resulted in higher subsequence scores, although the actualsequence can be tailored to reduce the degree of repetitiveness toresult in lower subsequence scores.

TABLE 34 Subsequence score calculations of polypeptide sequences SEQ SeqID Name Amino Acid Sequence NO: Score J288GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE 655 33.3GGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE G K288GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEG 656 46.9GGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE G L288SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESS 657 50.0SESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSES Y288GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEG 658 26.8SEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE Q576GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGG 659 18.5GEGGKPEGGKPEGEGKPGGGEGKPGGKPEGGGGKPEGEGKPGGGGGKPGGKPEGEGKPGGGEGGKPEGKPGEGGEGKPGGKPEGGGEGKPGGGKPGEGGKPGEGKPGGGEGGKPEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGEGGEGKPGGGKPEGEGKPGGGKPGGGEGGKPEGEGKPGGKPEGGGEGKPGGKPEGGGKPEGGGEGKPGGGKPGEGGKPGEGEGKPGGKPEGEGKPGGEGGGKPEGKPGGGEGGKPEGGKPGEGGKPEGGKPGEGGEGKPGGGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGGKPEGGKPEGGGEGKPGGGKPEGEGKPGGGEGKPGGKPEGGGGKPGEGGKPEGGKPGGEGGGKPEGEGKPGGKPGEGGGGKPGGKPEGEGKPGEGGEGKPGGKPEGGGEGKPGGKPEGGGEGKPGGGKPGEGGKPEGGGKPGEGGKPGEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGGEGGGKPEGGKPGEGGKP EG U576GEGKPGGKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKP 660 18.1GGKPGEGGKPEGGSGGKPGGGGKPGGKPGGEGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPEGGSGGKPGGKPEGGSGGKPGGSGKPGGKPGEGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPGGKPGEGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPGSGGEGKPGGKPEGGSGGKPGGGKPGGEGKPGSGGKPGEGGKPGSGGGKPGGKPGGEGEGKPGGKPGEGGKPGGEGSGKPGGGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPEGGGGKPEGSGKPGGGGKPEGSGKPGGGKPEGGSGGKPGGSGKPGGKPGEGGGKPEGSGKPGGGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGGEGSGKPGGKPGSGEGGKPGGKPGEGSGGKPGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPG GKPGEGGKPGGEGSGKPGGSGKPG W576GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPG 661 23.4SGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGGKPGGSGGKPGGGSGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGGSGGKPGKPGSGGGSGKPGKPGSGGSGGKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGKPGSGKPGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGGSGGKPGGSGGKPGKPGSGGGSGKPGKPGSGGGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGGKPGKPGSGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGSGKPGSGKPGGGSGGKPGKPGSGGSGKPGSGKPGSGGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGSGGKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGG Y576GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGS 662 15.7GEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEG SEGEGSEGSGEGEGSGEGSE AD576GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSE 663 13.6GGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS AE576AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 664  6.1STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AF540GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESP 665  8.8GPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AF504GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS 666  7.0SSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSP AE864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS 667  6.1TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAP AF864GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGT 668  7.5APGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATG SPGSSTPSGATGSP AG868GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS 669  7.5GTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSP AM875GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAES 670  4.5PGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAP AM1318GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSTAES 671  4.5PGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAP

Example 44: Calculation of TEPITOPE Scores

TEPITOPE scores of 9mer peptide sequence can be calculated by addingpocket potentials as described by Sturniolo [Sturniolo, T., et al.(1999) Nat Biotechnol, 17: 555]. In the present Example, separateTepitope scores were calculated for individual HLA alleles. Table 35shows as an example the pocket potentials for HLA*0101B, which occurs inhigh frequency in the Caucasian population. To calculate the TEPITOPEscore of a peptide with sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, thecorresponding individual pocket potentials in Table 35 were added. TheHLA*0101B score of a 9mer peptide with the sequence FDKLPRTSG (SEQ IDNO: 672) is the sum of 0, −1.3, 0, 0.9, 0, −1.8, 0.09, 0, 0.

To evaluate the TEPITOPE scores for long peptides one can repeat theprocess for all 9mer subsequences of the sequences. This process can berepeated for the proteins encoded by other HLA alleles. Tables 36-39give pocket potentials for the protein products of HLA alleles thatoccur with high frequency in the Caucasian population.

TEPITOPE scores calculated by this method range from approximately −10to +10. However, 9mer peptides that lack a hydrophobic amino acid(FKLMVWY (SEQ ID NO: 673)) in P1 position have calculated TEPITOPEscores in the range of −1009 to −989. This value is biologicallymeaningless and reflects the fact that a hydrophobic amino acid servesas an anchor residue for HLA binding and peptides lacking a hydrophobicresidue in P1 are considered non binders to HLA. Because most XTENsequences lack hydrophobic residues, all combinations of 9mersubsequences will have TEPITOPEs in the range in the range of −1009 to−989. This method confirms that XTEN polypeptides may have few or nopredicted T-cell epitopes.

TABLE 35 Pocket potential for HLA*0101B allele. Amino Acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −2.4 — −2.7 −2 — −1.9 E −999 0.1 −1.2 −0.4 — −2.4 −0.6 — −1.9 F 00.8 0.8 0.08 — −2.1 0.3 — −0.4 G −999 0.5 0.2 −0.7 — −0.3 −1.1 — −0.8 H−999 0.8 0.2 −0.7 — −2.2 0.1 — −1.1 I −1 1.1 1.5 0.5 — −1.9 0.6 — 0.7 K−999 1.1 0 −2.1 — −2 −0.2 — −1.7 L −1 1 1 0.9 — −2 0.3 — 0.5 M −1 1.11.4 0.8 — −1.8 0.09 — 0.08 N −999 0.8 0.5 0.04 — −1.1 0.1 — −1.2 P −999−0.5 0.3 −1.9 — −0.2 0.07 — −1.1 Q −999 1.2 0 0.1 — −1.8 0.2 — −1.6 R−999 2.2 0.7 −2.1 — −1.8 0.09 — −1 S −999 −0.3 0.2 −0.7 — −0.6 −0.2 —−0.3 T −999 0 0 −1 — −1.2 0.09 — −0.2 V −1 2.1 0.5 −0.1 — −1.1 0.7 — 0.3W 0 −0.1 0 −1.8 — −2.4 −0.1 — −1.4 Y 0 0.9 0.8 −1.1 — −2 0.5 — −0.9

TABLE 36 Pocket potential for HLA*0301B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 2.3 — −2.4 −0.6 — −0.6 E −999 0.1 −1.2 −1 — −1.4 −0.2 — −0.3 F −10.8 0.8 −1 — −1.4 0.5 — 0.9 G −999 0.5 0.2 0.5 — −0.7 0.1 — 0.4 H −9990.8 0.2 0 — −0.1 −0.8 — −0.5 I 0 1.1 1.5 0.5 — 0.7 0.4 — 0.6 K −999 1.10 −1 — 1.3 −0.9 — −0.2 L 0 1 1 0 — 0.2 0.2 — −0 M 0 1.1 1.4 0 — −0.9 1.1— 1.1 N −999 0.8 0.5 0.2 — −0.6 −0.1 — −0.6 P −999 −0.5 0.3 −1 — 0.5 0.7— −0.3 Q −999 1.2 0 0 — −0.3 −0.1 — −0.2 R −999 2.2 0.7 −1 — 1 −0.9 —0.5 S −999 −0.3 0.2 0.7 — −0.1 0.07 — 1.1 T −999 0 0 −1 — 0.8 −0.1 —−0.5 V 0 2.1 0.5 0 — 1.2 0.2 — 0.3 W −1 −0.1 0 −1 — −1.4 −0.6 — −1 Y −10.9 0.8 −1 — −1.4 −0.1 — 0.3

TABLE 37 Pocket potential for HLA*0401B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 1.4 — −1.1 −0.3 — −1.7 E −999 0.1 −1.2 1.5 — −2.4 0.2 — −1.7 F 00.8 0.8 −0.9 — −1.1 −1 — −1 G −999 0.5 0.2 −1.6 — −1.5 −1.3 — −1 H −9990.8 0.2 1.1 — −1.4 0 — 0.08 I −1 1.1 1.5 0.8 — −0.1 0.08 — −0.3 K −9991.1 0 −1.7 — −2.4 −0.3 — −0.3 L −1 1 1 0.8 — −1.1 0.7 — −1 M −1 1.1 1.40.9 — −1.1 0.8 — −0.4 N −999 0.8 0.5 0.9 — 1.3 0.6 — −1.4 P −999 −0.50.3 −1.6 — 0 −0.7 — −1.3 Q −999 1.2 0 0.8 — −1.5 0 — 0.5 R −999 2.2 0.7−1.9 — −2.4 −1.2 — −1 S −999 −0.3 0.2 0.8 — 1 −0.2 — 0.7 T −999 0 0 0.7— 1.9 −0.1 — −1.2 V −1 2.1 0.5 −0.9 — 0.9 0.08 — −0.7 W 0 −0.1 0 −1.2 —−1 −1.4 — −1 Y 0 0.9 0.8 −1.6 — −1.5 −1.2 — −1

TABLE 38 Pocket potential for HLA*0701B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −1.6 — −2.5 −1.3 — −1.2 E −999 0.1 −1.2 −1.4 — −2.5 0.9 — −0.3 F 00.8 0.8 0.2 — −0.8 2.1 — 2.1 G −999 0.5 0.2 −1.1 — −0.6 0 — −0.6 H −9990.8 0.2 0.1 — −0.8 0.9 — −0.2 I −1 1.1 1.5 1.1 — −0.5 2.4 — 3.4 K −9991.1 0 −1.3 — −1.1 0.5 — −1.1 L −1 1 1 −0.8 — −0.9 2.2 — 3.4 M −1 1.1 1.4−0.4 — −0.8 1.8 — 2 N −999 0.8 0.5 −1.1 — −0.6 1.4 — −0.5 P −999 −0.50.3 −1.2 — −0.5 −0.2 — −0.6 Q −999 1.2 0 −1.5 — −1.1 1.1 — −0.9 R −9992.2 0.7 −1.1 — −1.1 0.7 — −0.8 S −999 −0.3 0.2 1.5 — 0.6 0.4 — −0.3 T−999 0 0 1.4 — −0.1 0.9 — 0.4 V −1 2.1 0.5 0.9 — 0.1 1.6 — 2 W 0 −0.1 0−1.1 — −0.9 1.4 — 0.8 Y 0 0.9 0.8 −0.9 — −1 1.7 — 1.1

TABLE 39 Pocket potential for HLA*1501B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −0.4 — −0.4 −0.7 — −1.9 E −999 0.1 −1.2 −0.6 — −1 −0.7 — −1.9 F −10.8 0.8 2.4 — −0.3 1.4 — −0.4 G −999 0.5 0.2 0 — 0.5 0 — −0.8 H −999 0.80.2 1.1 — −0.5 0.6 — −1.1 I 0 1.1 1.5 0.6 — 0.05 1.5 — 0.7 K −999 1.1 0−0.7 — −0.3 −0.3 — −1.7 L 0 1 1 0.5 — 0.2 1.9 — 0.5 M 0 1.1 1.4 1 — 0.11.7 — 0.08 N −999 0.8 0.5 −0.2 — 0.7 0.7 — −1.2 P −999 −0.5 0.3 −0.3 —−0.2 0.3 — −1.1 Q −999 1.2 0 −0.8 — −0.8 −0.3 — −1.6 R −999 2.2 0.7 0.2— 1 −0.5 — −1 S −999 −0.3 0.2 −0.3 — 0.6 0.3 — −0.3 T −999 0 0 −0.3 — −00.2 — −0.2 V 0 2.1 0.5 0.2 — −0.3 0.3 — 0.3 W −1 −0.1 0 0.4 — −0.4 0.6 —−1.4 Y −1 0.9 0.8 2.5 — 0.4 0.7 — −0.9

TABLE 40 Exemplary Biological Activity, Exemplary Assays and PreferredIndications Biologically Exemplary Active Protein Biological ActivityActivity Assay Preferred Indication: Factor IX Coagulation factor IX isFactor IX clotting Hemophilia B; bleeding; (Coagulation a vitaminK-dependent activity: Valder Factor IX deficiency; factor IX factor thatcirculates in R. et al., 2001 Christmas disease; (human); the blood asan inactive “Posttranslational bleeding episodes in Factor IX zymogen.Factor IX is modifications of patients with factor Complex; converted toan active recombinant myotube- VIII inhibitor or Christmas factor; formby factor XIa, synthesized human Factor VII deficiency plasma whichexcises the factor IX” Blood thromboplastin activation peptide and 97:130-138. Activated component (PTC); thus generates a heavy partialthromboplastin prothrombin chain and a light chain time: Rao LV,Activation complex held together by one or of human factor VIIconcentrate more disulfide bonds. In during clotting in vitro (PCC); theblood coagulation Blood. 1985; 65(1): 218-26; Nonacog alpha; cascade,activated factor Park CH, A diagnostic MONONINE; IX activates factor Xto challenge: mild hemophilia ALPHANINE-SD; its active form through Bwith normal activated BEBULIN; interactions with Ca + 2 partialthromboplastin PROPLEX-T; ions, membrane time. Blood Coagul KONYNE;phospholipids, and factor Fibrinolysis. 2010 PROFILNINE SD; VIII.Alterations of this Jun; 21(4): 368-71. BeneFIX; gene, including pointIMMUNINE VH) mutations, insertions and deletions, cause factor IXdeficiency, which is a recessive X-linked disorder, also calledhemophilia B or Christmas disease. Factor VII Coagulation factor VII isCoagulation Assay Bleeding Disorders; (Coagulation a vitamin K-dependentusing Prothrombin Coronary Restenosis; Factor VII; factor essential forClotting Time Hemophilia A and B; Active-site hemostasis. This(Belaaouaj AA et Liver Disorders; inactivated factor circulates in theal., J. Biol. Chem. Thrombosis; Vascular factor VII blood in a zymogenform, 275: 27123-8, Restenosis; Surgery- (DEGR-VIIa/ and is converted toan 2000; Diaz-Collier related hemorrhagic FFR-VIIa); active form byeither JA et al., Thromb episodes Eptacog alfa; factor IXa, factor Xa,Haemost 71: 339- Coagulation factor XIIa, or thrombin 46,1994). Factorby minor proteolysis. Upon Vila; Novoseven; activation of the factorNiaStase; VII, a heavy chain Novostase; containing a catalyticMONOCLATE-P) domain and a light chain containing 2 EGF-like domains aregenerated, and two chains are held together by a disulfide bond. In thepresence of factor III and calcium ions, the activated factor thenfurther activates the coagulation cascade by converting factor IX tofactor IXa and/or factor X to factor Xa. Defects in this gene can causecoagulopathy.

TABLE 41 Exemplary CFXTEN comprising CF and single XTEN SEQ SEQ CFXTENAmino Acid ID DNA Nucleotide ID Name* Sequence NO: Sequence NO: FVII-ANAFLEELRPGSLE 674 GCCAACGCGTTCCTGGAGGAGC 675 AE288 RECKEEQCSFEEATACGGCCGGGCTCCCTGGAGAG REIFKDAERTKLF GGAGTGCAAGGAGGAGCAGTG WISYSDGDQCASSCTCCTTCGAGGAGGCCCGGGAG PCQNGGSCKDQLQ ATCTTCAAGGACGCGGAGAGGASYICFCLPAFEGRN CGAAGCTGTTCTGGATTTCTTAC CETHKDDQLICVNAGTGATGGGGACCAGTGTGCCT ENGGCEQYCSDHT CAAGTCCATGCCAGAATGGGGGGTKRSCRCHEGYS CTCCTGCAAGGACCAGCTCCAG LLADGVSCTPTVETCCTATATCTGCTTCTGCCTCCC YPCGKIPILEKRNA TGCCTTCGAGGGCCGGAACTGTSKPQGRIVGGKVC GAGACGCACAAGGATGACCAG PKGECPWQVLLLV CTGATCTGTGTGAACGAGAACGNGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAG WVVSAAHCFDKIKTGACCACACGGGCACCAAGCGC NWRNLIAVLGEHD TCCTGTCGGTGCCACGAGGGGTLSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGT AQVIIPSTYVPGTTGTCCTGCACACCCACAGTTGAA NHDIALLRLHQPV TATCCATGTGGAAAAATACCTAVLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAG RTFSERTLAFVRFSCAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAA ATALELMVLNVPRGGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCA VGDSPNITEYMFCGTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKG AACACCATCTGGGTGGTCTCCGDSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQG CAAGAACTGGAGGAACCTGATCCATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACC SQYIEWLQKLMRS TCAGCGAGCACGACGGGGATGEPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGC GTSESATPESGPGSAGGTCATCATCCCCAGCACGTA EPATSGSETPGTSE CGTCCCGGGCACCACCAACCACSATPESGPGSEPAT GACATCGCGCTGCTCCGCCTGC SGSETPGTSESATPACCAGCCCGTGGTCCTCACTGA ESGPGTSTEPSEGS CCATGTGGTGCCCCTCTGCCTGAPGSPAGSPTSTEE CCCGAACGGACGTTCTCTGAGA GTSESATPESGPGSGGACGCTGGCCTTCGTGCGCTT EPATSGSETPGTSE CTCATTGGTCAGCGGCTGGGGCSATPESGPGSPAGS CAGCTGCTGGACCGTGGCGCCA PTSTEEGSPAGSPTCGGCCCTGGAGCTCATGGTCCT STEEGTSTEPSEGS CAACGTGCCCCGGCTGATGACCAPGTSESATPESGP CAGGACTGCCTGCAGCAGTCAC GTSESATPESGPGTGGAAGGTGGGAGACTCCCCAA SESATPESGPGSEP ATATCACGGAGTACATGTTCTGATSGSETPGSEPAT TGCCGGCTACTCGGATGGCAGC SGSETPGSPAGSPTAAGGACTCCTGCAAGGGGGAC STEEGTSTEPSEGS AGTGGAGGCCCACATGCCACCCAPGTSTEPSEGSAP ACTACCGGGGCACGTGGTACCT GSEPATSGSETPGTGACGGGCATCGTCAGCTGGGGC SESATPESGPGTST CAGGGCTGCGCAACCGTGGGCC EPSEGSAPACTTTGGGGTGTACACCAGGGT CTCCCAGTACATCGAGTGGCTG CAAAAGCTCATGCGCTCAGAGCCACGCCCAGGAGTCCTCCTGCG AGCCCCATTTCCCGGTGGTACC TCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC TACCTCCGGCTCTGAGACTCCA GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGA ACCTGCAACCTCTGGCTCTGAA ACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGG TACTTCTACTGAACCGTCCGAG GGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA AGAAGGTACCTCTGAAAGCGCA ACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTC TGAAACCCCAGGTACTTCTGAA AGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCC GACTTCCACCGAGGAAGGTAGC CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGA ACCTTCCGAGGGCAGCGCACCA GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTC TGAAAGCGCTACTCCTGAATCC GGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGG TAGCGAACCGGCTACTTCTGGT TCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAAC TCCAGGTAGCCCAGCAGGCTCT CCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGG CAGCGCACCAGGTACCTCTACT GAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTC TGGCTCTGAAACCCCAGGTACC TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGA ACCGTCCGAGGGCAGCGCACCA FVII- ANAFLEELRPGSLE 676GCCAACGCGTTCCTGGAGGAGC 677 AE864 RECKEEQCSFEEA TACGGCCGGGCTCCCTGGAGAGREIFKDAERTKLF GGAGTGCAAGGAGGAGCAGTG WISYSDGDQCASS CTCCTTCGAGGAGGCCCGGGAGPCQNGGSCKDQLQ ATCTTCAAGGACGCGGAGAGGA SYICFCLPAFEGRNCGAAGCTGTTCTGGATTTCTTAC CETHKDDQLICVN AGTGATGGGGACCAGTGTGCCTENGGCEQYCSDHT CAAGTCCATGCCAGAATGGGGG GTKRSCRCHEGYSCTCCTGCAAGGACCAGCTCCAG LLADGVSCTPTVE TCCTATATCTGCTTCTGCCTCCCYPCGKIPILEKRNA TGCCTTCGAGGGCCGGAACTGT SKPQGRIVGGKVCGAGACGCACAAGGATGACCAG PKGECPWQVLLLV CTGATCTGTGTGAACGAGAACGNGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAG WVVSAAHCFDKIKTGACCACACGGGCACCAAGCGC NWRNLIAVLGEHD TCCTGTCGGTGCCACGAGGGGTLSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGT AQVIIPSTYVPGTTGTCCTGCACACCCACAGTTGAA NHDIALLRLHQPV TATCCATGTGGAAAAATACCTAVLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAG RTFSERTLAFVRFSCAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAA ATALELMVLNVPRGGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCA VGDSPNITEYMFCGTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKG AACACCATCTGGGTGGTCTCCGDSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQG CAAGAACTGGAGGAACCTGATCCATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACC SQYIEWLQKLMRS TCAGCGAGCACGACGGGGATGEPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGC GSPAGSPTSTEEGTAGGTCATCATCCCCAGCACGTA SESATPESGPGTST CGTCCCGGGCACCACCAACCACEPSEGSAPGSPAGS GACATCGCGCTGCTCCGCCTGC PTSTEEGTSTEPSEACCAGCCCGTGGTCCTCACTGA GSAPGTSTEPSEGS CCATGTGGTGCCCCTCTGCCTGAPGTSESATPESGP CCCGAACGGACGTTCTCTGAGA GSEPATSGSETPGSGGACGCTGGCCTTCGTGCGCTT EPATSGSETPGSPA CTCATTGGTCAGCGGCTGGGGCGSPTSTEEGTSESA CAGCTGCTGGACCGTGGCGCCA TPESGPGTSTEPSECGGCCCTGGAGCTCATGGTCCT GSAPGTSTEPSEGS CAACGTGCCCCGGCTGATGACCAPGSPAGSPTSTEE CAGGACTGCCTGCAGCAGTCAC GTSTEPSEGSAPGTGGAAGGTGGGAGACTCCCCAA STEPSEGSAPGTSE ATATCACGGAGTACATGTTCTGSATPESGPGTSTEP TGCCGGCTACTCGGATGGCAGC SEGSAPGTSESATPAAGGACTCCTGCAAGGGGGAC ESGPGSEPATSGSE AGTGGAGGCCCACATGCCACCCTPGTSTEPSEGSAP ACTACCGGGGCACGTGGTACCT GTSTEPSEGSAPGTGACGGGCATCGTCAGCTGGGGC SESATPESGPGTSE CAGGGCTGCGCAACCGTGGGCCSATPESGPGSPAGS ACTTTGGGGTGTACACCAGGGT PTSTEEGTSESATPCTCCCAGTACATCGAGTGGCTG ESGPGSEPATSGSE CAAAAGCTCATGCGCTCAGAGCTPGTSESATPESGP CACGCCCAGGAGTCCTCCTGCG GTSTEPSEGSAPGTAGCCCCATTTCCCGGTGGTAGC STEPSEGSAPGTST CCGGCTGGCTCTCCTACCTCTACEPSEGSAPGTSTEP TGAGGAAGGTACTTCTGAAAGC SEGSAPGTSTEPSEGCTACTCCTGAGTCTGGTCCAG GSAPGTSTEPSEGS GTACCTCTACTGAACCGTCCGAAPGSPAGSPTSTEE AGGTAGCGCTCCAGGTAGCCCA GTSTEPSEGSAPGTGCAGGCTCTCCGACTTCCACTG SESATPESGPGSEP AGGAAGGTACTTCTACTGAACCATSGSETPGTSESA TTCCGAAGGCAGCGCACCAGGT TPESGPGSEPATSGACCTCTACTGAACCTTCTGAGG SETPGTSESATPES GCAGCGCTCCAGGTACTTCTGAGPGTSTEPSEGSAP AAGCGCTACCCCGGAATCTGGC GTSESATPESGPGSCCAGGTAGCGAACCGGCTACTT PAGSPTSTEEGSPA CTGGTTCTGAAACCCCAGGTAGGSPTSTEEGSPAGS CGAACCGGCTACCTCCGGTTCT PTSTEEGTSESATPGAAACTCCAGGTAGCCCGGCAG ESGPGTSTEPSEGS GCTCTCCGACCTCTACTGAGGAAPGTSESATPESGP AGGTACTTCTGAAAGCGCAACC GSEPATSGSETPGTCCGGAGTCCGGCCCAGGTACCT SESATPESGPGSEP CTACCGAACCGTCTGAGGGCAGATSGSETPGTSESA CGCACCAGGTACTTCTACCGAA TPESGPGTSTEPSECCGTCCGAGGGTAGCGCACCAG GSAPGSPAGSPTST GTAGCCCAGCAGGTTCTCCTACEEGTSESATPESGP CTCCACCGAGGAAGGTACTTCT GSEPATSGSETPGTACCGAACCGTCCGAGGGTAGCG SESATPESGPGSPA CACCAGGTACCTCTACTGAACCGSPTSTEEGSPAGS TTCTGAGGGCAGCGCTCCAGGT PTSTEEGTSTEPSEACTTCTGAAAGCGCTACCCCGG GSAPGTSESATPES AGTCCGGTCCAGGTACTTCTACGPGTSESATPESGP TGAACCGTCCGAAGGTAGCGCA GTSESATPESGPGSCCAGGTACTTCTGAAAGCGCAA EPATSGSETPGSEP CCCCTGAATCCGGTCCAGGTAGATSGSETPGSPAGS CGAACCGGCTACTTCTGGCTCT PTSTEEGTSTEPSEGAGACTCCAGGTACTTCTACCG GSAPGTSTEPSEGS AACCGTCCGAAGGTAGCGCACCAPGSEPATSGSETP AGGTACTTCTACTGAACCGTCT GTSESATPESGPGTGAAGGTAGCGCACCAGGTACTT STEPSEGSAP CTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGC GCAACCCCGGAGTCCGGCCCAG GTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT GAAAGCGCAACCCCTGAATCCG GCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGT ACCTCTGAAAGCGCTACTCCGG AGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCT CCAGGTACTTCTACTGAACCGT CCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGC AGCGCTCCAGGTACCTCTACTG AACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCT GAAGGTAGCGCACCAGGTACTT CTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGT TCTCCTACCTCCACCGAGGAAG GTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCT GAAAGCGCAACTCCTGAGTCTG GCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGT ACCTCTGAAAGCGCAACCCCGG AATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACC CCAGGTACCTCTGAAAGCGCTA CTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC AGCGCACCAGGTACTTCTGAAA GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCG ACTTCCACCGAGGAAGGTAGCC CGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGC TCTCCGACCTCTACTGAGGAAG GTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCT ACCGAACCGTCTGAGGGCAGCG CACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGT AGCGAACCTGCTACCTCCGGCT CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGT CCAGGTAGCGAACCTGCAACCT CTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA TCTGGCCCAGGTACTTCTACTG AACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCA ACCTCCACCGAAGAAGGTACCT CTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA ACCTCCGGTTCTGAAACCCCAG GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCG GCTGGCTCTCCGACTTCCACCG AGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGT ACTTCTACCGAACCTTCCGAGG GCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGC CCAGGTACTTCTGAAAGCGCTA CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAA TCTGGCCCAGGTAGCGAACCGG CTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCC GGTTCTGAAACTCCAGGTAGCC CAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAA CCTTCCGAAGGCAGCGCACCAG GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAA CCTGCAACCTCTGGCTCTGAAA CCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGT ACTTCTACTGAACCGTCCGAGG GCAGCGCACCA FVII-ANAFLEELRPGSLE 678 GCCAACGCGTTCCTGGAGGAGC 679 AF864 RECKEEQCSFEEATACGGCCGGGCTCCCTGGAGAG REIFKDAERTKLF GGAGTGCAAGGAGGAGCAGTG WISYSDGDQCASSCTCCTTCGAGGAGGCCCGGGAG PCQNGGSCKDQLQ ATCTTCAAGGACGCGGAGAGGASYICFCLPAFEGRN CGAAGCTGTTCTGGATTTCTTAC CETHKDDQLICVNAGTGATGGGGACCAGTGTGCCT ENGGCEQYCSDHT CAAGTCCATGCCAGAATGGGGGGTKRSCRCHEGYS CTCCTGCAAGGACCAGCTCCAG LLADGVSCTPTVETCCTATATCTGCTTCTGCCTCCC YPCGKIPILEKRNA TGCCTTCGAGGGCCGGAACTGTSKPQGRIVGGKVC GAGACGCACAAGGATGACCAG PKGECPWQVLLLV CTGATCTGTGTGAACGAGAACGNGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAG WVVSAAHCFDKIKTGACCACACGGGCACCAAGCGC NWRNLIAVLGEHD TCCTGTCGGTGCCACGAGGGGTLSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGT AQVIIPSTYVPGTTGTCCTGCACACCCACAGTTGAA NHDIALLRLHQPV TATCCATGTGGAAAAATACCTAVLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAG RTFSERTLAFVRFSCAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAA ATALELMVLNVPRGGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCA VGDSPNITEYMFCGTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKG AACACCATCTGGGTGGTCTCCGDSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQG CAAGAACTGGAGGAACCTGATCCATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACC SQYIEWLQKLMRS TCAGCGAGCACGACGGGGATGEPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGC GSTSESPSGTAPGTAGGTCATCATCCCCAGCACGTA SPSGESSTAPGSTS CGTCCCGGGCACCACCAACCACESPSGTAPGSTSES GACATCGCGCTGCTCCGCCTGC PSGTAPGTSTPESGACCAGCCCGTGGTCCTCACTGA SASPGTSTPESGSA CCATGTGGTGCCCCTCTGCCTGSPGSTSESPSGTAP CCCGAACGGACGTTCTCTGAGA GSTSESPSGTAPGTGGACGCTGGCCTTCGTGCGCTT SPSGESSTAPGSTS CTCATTGGTCAGCGGCTGGGGCESPSGTAPGTSPSG CAGCTGCTGGACCGTGGCGCCA ESSTAPGTSPSGESCGGCCCTGGAGCTCATGGTCCT STAPGSTSSTAESP CAACGTGCCCCGGCTGATGACCGPGTSPSGESSTAP CAGGACTGCCTGCAGCAGTCAC GTSPSGESSTAPGSGGAAGGTGGGAGACTCCCCAA TSSTAESPGPGTST ATATCACGGAGTACATGTTCTGPESGSASPGTSTPE TGCCGGCTACTCGGATGGCAGC SGSASPGSTSESPSAAGGACTCCTGCAAGGGGGAC GTAPGSTSESPSGT AGTGGAGGCCCACATGCCACCCAPGTSTPESGSASP ACTACCGGGGCACGTGGTACCT GSTSSTAESPGPGTGACGGGCATCGTCAGCTGGGGC STPESGSASPGSTS CAGGGCTGCGCAACCGTGGGCCESPSGTAPGTSPSG ACTTTGGGGTGTACACCAGGGT ESSTAPGSTSSTAECTCCCAGTACATCGAGTGGCTG SPGPGTSPSGESST CAAAAGCTCATGCGCTCAGAGCAPGTSTPESGSASP CACGCCCAGGAGTCCTCCTGCG GSTSSTAESPGPGSAGCCCCATTTCCCGGTGGTTCT TSSTAESPGPGSTS ACCAGCGAATCTCCTTCTGGCASTAESPGPGSTSST CCGCTCCAGGTACCTCTCCTAG AESPGPGTSPSGESCGGCGAATCTTCTACCGCTCCA STAPGSTSESPSGT GGTTCTACTAGCGAATCTCCTTCAPGSTSESPSGTAP TGGCACTGCACCAGGTTCTACT GTSTPESGPXXXGAGCGAATCCCCGTCTGGTACTG ASASGAPSTXXXX CTCCAGGTACTTCTACTCCTGASESPSGTAPGSTSE AAGCGGTTCCGCTTCTCCAGGT SPSGTAPGSTSESPACCTCTACTCCGGAAAGCGGTT SGTAPGSTSESPSG CTGCATCTCCAGGTTCTACCAGTAPGSTSESPSGTA CGAATCTCCTTCTGGCACCGCT PGSTSESPSGTAPGCCAGGTTCTACTAGCGAATCCC TSTPESGSASPGTS CGTCTGGTACCGCACCAGGTACPSGESSTAPGTSPS TTCTCCTAGCGGCGAATCTTCTA GESSTAPGSTSSTACCGCACCAGGTTCTACTAGCGA ESPGPGTSPSGESS ATCTCCGTCTGGCACTGCTCCATAPGTSTPESGSAS GGTACTTCTCCTAGCGGTGAAT PGSTSESPSGTAPGCTTCTACCGCTCCAGGTACTTCC STSESPSGTAPGTS CCTAGCGGCGAATCTTCTACCGPSGESSTAPGSTSE CTCCAGGTTCTACTAGCTCTACT SPSGTAPGTSTPESGCAGAATCTCCGGGCCCAGGTA GSASPGTSTPESGS CCTCTCCTAGCGGTGAATCTTCTASPGSTSESPSGTA ACCGCTCCAGGTACTTCTCCGA PGTSTPESGSASPGGCGGTGAATCTTCTACCGCTCC STSSTAESPGPGST AGGTTCTACTAGCTCTACTGCASESPSGTAPGSTSE GAATCTCCTGGCCCAGGTACCT SPSGTAPGTSPSGECTACTCCGGAAAGCGGCTCTGC SSTAPGSTSSTAES ATCTCCAGGTACTTCTACCCCTGPGPGTSPSGESSTA AAAGCGGTTCTGCATCTCCAGG PGTSTPESGSASPGTTCTACTAGCGAATCTCCTTCTG TSPSGESSTAPGTS GCACTGCACCAGGTTCTACCAGPSGESSTAPGTSPS CGAATCTCCGTCTGGCACTGCA GESSTAPGSTSSTACCAGGTACCTCTACCCCTGAAA ESPGPGSTSSTAES GCGGTTCCGCTTCTCCAGGTTCTPGPGTSPSGESSTA ACCAGCTCTACCGCAGAATCTC PGSSPSASTGTGPGCTGGTCCAGGTACCTCTACTCC SSTPSGATGSPGSS GGAAAGCGGCTCTGCATCTCCA TPSGATGSPGGTTCTACTAGCGAATCTCCTTC TGGCACTGCACCAGGTACTTCT CCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTAC CGCTGAATCTCCGGGCCCAGGT ACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACT CCTGAAAGCGGTTCTGCATCTC CAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCT ACTAGCTCTACTGCTGAATCTC CTGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCA GGTTCTACCAGCTCTACTGCTG AATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACT GCACCAGGTTCTACTAGCGAAT CTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCT GGCACTGCACCAGGTACCTCTA CCCCTGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGCAAGCGG CGCGCCAAGCACGGGAXXXXX XXXTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCG AATCCCCGTCTGGTACTGCTCC AGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTA CTAGCGAATCTCCTTCTGGTAC CGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAG GTTCTACCAGCGAATCTCCTTCT GGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATC TCCAGGTACTTCTCCTAGCGGT GAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCT ACTGCTCCAGGTTCTACCAGCT CTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAA TCTTCTACTGCACCAGGTACTTC TACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAAT CTCCTTCTGGCACCGCTCCAGG TTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTC CTAGCGGCGAATCTTCTACCGC ACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTA CTTCTACCCCGGAAAGCGGCTC TGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCC AGGTTCTACTAGCGAATCTCCT TCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGC TTCTCCAGGTTCCACTAGCTCTA CCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTG GCACCGCTCCAGGTTCTACTAG CGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCG AATCTTCTACCGCACCAGGTTC TACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGA GCGGTGAATCTTCTACTGCACC AGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTC CCCTAGCGGCGAATCTTCTACT GCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG TACCTCCCCTAGCGGTGAATCT TCTACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGG TCCAGGTTCTACCAGCTCTACT GCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTC TACTGCACCAGGTTCTAGCCCT TCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGG TGCAACTGGCTCTCCAGGTAGC TCTACTCCGTCTGGTGCAACCGGCTCCCCA FVII- ANAFLEELRPGSLE 680 GCCAACGCGTTCCTGGAGGAGC 681 AG864RECKEEQCSFEEA TACGGCCGGGCTCCCTGGAGAG REIFKDAERTKLF GGAGTGCAAGGAGGAGCAGTGWISYSDGDQCASS CTCCTTCGAGGAGGCCCGGGAG PCQNGGSCKDQLQATCTTCAAGGACGCGGAGAGGA SYICFCLPAFEGRN CGAAGCTGTTCTGGATTTCTTACCETHKDDQLICVN AGTGATGGGGACCAGTGTGCCT ENGGCEQYCSDHTCAAGTCCATGCCAGAATGGGGG GTKRSCRCHEGYS CTCCTGCAAGGACCAGCTCCAGLLADGVSCTPTVE TCCTATATCTGCTTCTGCCTCCC YPCGKIPILEKRNATGCCTTCGAGGGCCGGAACTGT SKPQGRIVGGKVC GAGACGCACAAGGATGACCAG PKGECPWQVLLLVCTGATCTGTGTGAACGAGAACG NGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAGWVVSAAHCFDKIK TGACCACACGGGCACCAAGCGC NWRNLIAVLGEHDTCCTGTCGGTGCCACGAGGGGT LSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGTAQVIIPSTYVPGTT GTCCTGCACACCCACAGTTGAA NHDIALLRLHQPVTATCCATGTGGAAAAATACCTA VLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAGRTFSERTLAFVRFS CAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAAATALELMVLNVPR GGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCAVGDSPNITEYMFC GTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKGAACACCATCTGGGTGGTCTCCG DSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQGCAAGAACTGGAGGAACCTGATC CATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACCSQYIEWLQKLMRS TCAGCGAGCACGACGGGGATG EPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGCGASPGTSSTGSPGS AGGTCATCATCCCCAGCACGTA SPSASTGTGPGSSPCGTCCCGGGCACCACCAACCAC SASTGTGPGTPGS GACATCGCGCTGCTCCGCCTGCGTASSSPGSSTPSG ACCAGCCCGTGGTCCTCACTGA ATGSPGSNPSASTGCCATGTGGTGCCCCTCTGCCTG TGPGASPGTSSTGS CCCGAACGGACGTTCTCTGAGAPGTPGSGTASSSPG GGACGCTGGCCTTCGTGCGCTT SSTPSGATGSPGTPCTCATTGGTCAGCGGCTGGGGC GSGTASSSPGASPG CAGCTGCTGGACCGTGGCGCCATSSTGSPGASPGTS CGGCCCTGGAGCTCATGGTCCT STGSPGTPGSGTASCAACGTGCCCCGGCTGATGACC SSPGSSTPSGATGS CAGGACTGCCTGCAGCAGTCACPGASPGTSSTGSPG GGAAGGTGGGAGACTCCCCAA TPGSGTASSSPGSSATATCACGGAGTACATGTTCTG TPSGATGSPGSNPS TGCCGGCTACTCGGATGGCAGCASTGTGPGSSPSAS AAGGACTCCTGCAAGGGGGAC TGTGPGSSTPSGATAGTGGAGGCCCACATGCCACCC GSPGSSTPSGATGS ACTACCGGGGCACGTGGTACCTPGASPGTSSTGSPG GACGGGCATCGTCAGCTGGGGC ASPGTSSTGSPGASCAGGGCTGCGCAACCGTGGGCC PGTSSTGSPGTPGS ACTTTGGGGTGTACACCAGGGTGTASSSPGASPGTS CTCCCAGTACATCGAGTGGCTG STGSPGASPGTSSTCAAAAGCTCATGCGCTCAGAGC GSPGASPGTSSTGS CACGCCCAGGAGTCCTCCTGCGPGSSPSASTGTGPG AGCCCCATTTCCCGGTGGTGCT TPGSGTASSSPGASTCCCCGGGCACCAGCTCTACTG PGTSSTGSPGASPG GTTCTCCAGGTTCTAGCCCGTCTTSSTGSPGASPGTS GCTTCTACTGGTACTGGTCCAG STGSPGSSTPSGATGTTCTAGCCCTTCTGCTTCCACT GSPGSSTPSGATGS GGTACTGGTCCAGGTACCCCGGPGASPGTSSTGSPG GTAGCGGTACCGCTTCTTCTTCT TPGSGTASSSPGSSCCAGGTAGCTCTACTCCGTCTG TPSGATGSPGSSTP GTGCTACCGGCTCTCCAGGTTCSGATGSPGSSTPSG TAACCCTTCTGCATCCACCGGT ATGSPGSSPSASTGACCGGCCCAGGTGCTTCTCCGG TGPGASPGTSSTGS GCACCAGCTCTACTGGTTCTCCPGASPGTSSTGSPG AGGTACCCCGGGCAGCGGTACC TPGSGTASSSPGASGCATCTTCTTCTCCAGGTAGCTC PGTSSTGSPGASPG TACTCCTTCTGGTGCAACTGGTTTSSTGSPGASPGTS CTCCAGGTACTCCTGGCAGCGG STGSPGASPGTSSTTACCGCTTCTTCTTCTCCAGGTG GSPGTPGSGTASSS CTTCTCCTGGTACTAGCTCTACTPGSSTPSGATGSPG GGTTCTCCAGGTGCTTCTCCGG TPGSGTASSSPGSSGCACTAGCTCTACTGGTTCTCC TPSGATGSPGTPGS AGGTACCCCGGGTAGCGGTACTGTASSSPGSSTPSG GCTTCTTCCTCTCCAGGTAGCTC ATGSPGSSTPSGATTACCCCTTCTGGTGCAACCGGC GSPGSSPSASTGTG TCTCCAGGTGCTTCTCCGGGCAPGSSPSASTGTGPG CCAGCTCTACCGGTTCTCCAGG ASPGTSSTGSPGTPTACCCCGGGTAGCGGTACCGCT GSGTASSSPGSSTP TCTTCTTCTCCAGGTAGCTCTACSGATGSPGSSPSAS TCCGTCTGGTGCTACCGGCTCTC TGTGPGSSPSASTGCAGGTTCTAACCCTTCTGCATCC TGPGASPGTSSTGS ACCGGTACCGGCCCAGGTTCTAPGASPGTSSTGSPG GCCCTTCTGCTTCCACCGGTACT SSTPSGATGSPGSSGGCCCAGGTAGCTCTACCCCTT PSASTGTGPGASPG CTGGTGCTACCGGCTCCCCAGGTSSTGSPGSSPSAS TAGCTCTACTCCTTCTGGTGCAA TGTGPGTPGSGTACTGGCTCTCCAGGTGCATCTCC SSSPGSSTPSGATG GGGCACTAGCTCTACTGGTTCTSPGSSTPSGATGSP CCAGGTGCATCCCCTGGCACTA GASPGTSSTGSPGCTCTACTGGTTCTCCAGGTGCT TCTCCTGGTACCAGCTCTACTG GTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG GTGCTTCTCCTGGTACTAGCTCT ACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCT CCAGGTGCTTCCCCGGGCACTA GCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTA CTGGCCCAGGTACTCCGGGCAG CGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAGCTC TACTGGTTCTCCAGGTGCATCC CCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACC AGCTCTACTGGTTCTCCAGGTA GCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACT CCTTCTGGTGCTACTGGCTCCCC AGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAGGTACCC CGGGCAGCGGTACCGCATCTTC CTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG GTAGCTCTACCCCGTCTGGTGC AACCGGCTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCT CCCCAGGTTCTAGCCCGTCTGC TTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA CTGGTTCTCCAGGTGCATCCCC GGGTACCAGCTCTACCGGTTCTCCAGGTACTCCTGGCAGCGGTA CTGCATCTTCCTCTCCAGGTGCT TCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGG CACTAGCTCTACTGGTTCTCCA GGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCT CCTGGTACCAGCTCTACTGGTT CTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTA GCTCTACTCCGTCTGGTGCTACC GGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA GGTAGCTCTACCCCGTCTGGTG CTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCC TCTCCAGGTAGCTCTACCCCTTC TGGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTA CTGGCTCCCCAGGTTCTAGCCC TTCTGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCAT CTACTGGTACTGGTCCAGGTGC ATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTCCTGGTA GCGGTACTGCTTCTTCTTCTCCA GGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCC CTTCTGCATCCACCGGTACCGG CCCAGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGC TTCTCCGGGTACTAGCTCTACTG GTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAG GTAGCTCTACTCCGTCTGGTGC AACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTG GTCCAGGTGCATCCCCTGGTAC CAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACCGG TACCGGTCCAGGTACCCCTGGC AGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACTCCGTCTGG TGCAACCGGTTCCCCAGGTAGC TCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGC ACCAGCTCTACCGGTTCTCCA FVII- ANAFLEELRPGSLE 682GCCAACGCGTTCCTGGAGGAGC 683 AM875 RECKEEQCSFEEA TACGGCCGGGCTCCCTGGAGAGREIFKDAERTKLF GGAGTGCAAGGAGGAGCAGTG WISYSDGDQCASS CTCCTTCGAGGAGGCCCGGGAGPCQNGGSCKDQLQ ATCTTCAAGGACGCGGAGAGGA SYICFCLPAFEGRNCGAAGCTGTTCTGGATTTCTTAC CETHKDDQLICVN AGTGATGGGGACCAGTGTGCCTENGGCEQYCSDHT CAAGTCCATGCCAGAATGGGGG GTKRSCRCHEGYSCTCCTGCAAGGACCAGCTCCAG LLADGVSCTPTVE TCCTATATCTGCTTCTGCCTCCCYPCGKIPILEKRNA TGCCTTCGAGGGCCGGAACTGT SKPQGRIVGGKVCGAGACGCACAAGGATGACCAG PKGECPWQVLLLV CTGATCTGTGTGAACGAGAACGNGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAG WVVSAAHCFDKIKTGACCACACGGGCACCAAGCGC NWRNLIAVLGEHD TCCTGTCGGTGCCACGAGGGGTLSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGT AQVIIPSTYVPGTTGTCCTGCACACCCACAGTTGAA NHDIALLRLHQPV TATCCATGTGGAAAAATACCTAVLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAG RTFSERTLAFVRFSCAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAA ATALELMVLNVPRGGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCA VGDSPNITEYMFCGTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKG AACACCATCTGGGTGGTCTCCGDSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQG CAAGAACTGGAGGAACCTGATCCATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACC SQYIEWLQKLMRS TCAGCGAGCACGACGGGGATGEPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGC GTSTEPSEGSAPGSAGGTCATCATCCCCAGCACGTA EPATSGSETPGSPA CGTCCCGGGCACCACCAACCACGSPTSTEEGSTSST GACATCGCGCTGCTCCGCCTGC AESPGPGTSTPESGACCAGCCCGTGGTCCTCACTGA SASPGSTSESPSGT CCATGTGGTGCCCCTCTGCCTGAPGSTSESPSGTAP CCCGAACGGACGTTCTCTGAGA GTSTPESGSASPGTGGACGCTGGCCTTCGTGCGCTT STPESGSASPGSEP CTCATTGGTCAGCGGCTGGGGCATSGSETPGTSESA CAGCTGCTGGACCGTGGCGCCA TPESGPGSPAGSPTCGGCCCTGGAGCTCATGGTCCT STEEGTSTEPSEGS CAACGTGCCCCGGCTGATGACCAPGTSESATPESGP CAGGACTGCCTGCAGCAGTCAC GTSTEPSEGSAPGTGGAAGGTGGGAGACTCCCCAA STEPSEGSAPGSPA ATATCACGGAGTACATGTTCTGGSPTSTEEGTSTEP TGCCGGCTACTCGGATGGCAGC SEGSAPGTSTEPSEAAGGACTCCTGCAAGGGGGAC GSAPGTSESATPES AGTGGAGGCCCACATGCCACCCGPGTSESATPESGP ACTACCGGGGCACGTGGTACCT GTSTEPSEGSAPGTGACGGGCATCGTCAGCTGGGGC STEPSEGSAPGTSE CAGGGCTGCGCAACCGTGGGCCSATPESGPGTSTEP ACTTTGGGGTGTACACCAGGGT SEGSAPGSEPATSGCTCCCAGTACATCGAGTGGCTG SETPGSPAGSPTST CAAAAGCTCATGCGCTCAGAGCEEGSSTPSGATGSP CACGCCCAGGAGTCCTCCTGCG GTPGSGTASSSPGSAGCCCCATTTCCCGGTGGTACT STPSGATGSPGTST TCTACTGAACCGTCTGAAGGCAEPSEGSAPGTSTEP GCGCACCAGGTAGCGAACCGGC SEGSAPGSEPATSGTACTTCCGGTTCTGAAACCCCA SETPGSPAGSPTST GGTAGCCCAGCAGGTTCTCCAAEEGSPAGSPTSTEE CTTCTACTGAAGAAGGTTCTAC GTSTEPSEGSAPGACAGCTCTACCGCAGAATCTCCT SASGAPSTGGTSES GGTCCAGGTACCTCTACTCCGGATPESGPGSPAGSP AAAGCGGCTCTGCATCTCCAGG TSTEEGSPAGSPTSTTCTACTAGCGAATCTCCTTCTG TEEGSTSSTAESPG GCACTGCACCAGGTTCTACTAGPGSTSESPSGTAPG CGAATCCCCGTCTGGTACTGCT TSPSGESSTAPGTPCCAGGTACTTCTACTCCTGAAA GSGTASSSPGSSTP GCGGTTCCGCTTCTCCAGGTACSGATGSPGSSPSAS CTCTACTCCGGAAAGCGGTTCT TGTGPGSEPATSGSGCATCTCCAGGTAGCGAACCGG ETPGTSESATPESG CAACCTCCGGCTCTGAAACCCCPGSEPATSGSETPG AGGTACCTCTGAAAGCGCTACT STSSTAESPGPGSTCCTGAATCCGGCCCAGGTAGCC SSTAESPGPGTSPS CGGCAGGTTCTCCGACTTCCACGESSTAPGSEPATS TGAGGAAGGTACCTCTACTGAA GSETPGSEPATSGSCCTTCTGAGGGCAGCGCTCCAG ETPGTSTEPSEGSA GTACTTCTGAAAGCGCTACCCCPGSTSSTAESPGPG GGAGTCCGGTCCAGGTACTTCT TSTPESGSASPGSTACTGAACCGTCCGAAGGTAGCG SESPSGTAPGTSTE CACCAGGTACTTCTACCGAACCPSEGSAPGTSTEPS GTCCGAGGGTAGCGCACCAGGT EGSAPGTSTEPSEGAGCCCAGCAGGTTCTCCTACCT SAPGSSTPSGATGS CCACCGAGGAAGGTACTTCTACPGSSPSASTGTGPG CGAACCGTCCGAGGGTAGCGCA ASPGTSSTGSPGSECCAGGTACTTCTACCGAACCTT PATSGSETPGTSES CCGAGGGCAGCGCACCAGGTACATPESGPGSPAGSP TTCTGAAAGCGCTACCCCTGAG TSTEEGSSTPSGATTCCGGCCCAGGTACTTCTGAAA GSPGSSPSASTGTG GCGCTACTCCTGAATCCGGTCCPGASPGTSSTGSPG AGGTACCTCTACTGAACCTTCC TSESATPESGPGTSGAAGGCAGCGCTCCAGGTACCT TEPSEGSAPGTSTE CTACCGAACCGTCCGAGGGCAG PSEGSAPCGCACCAGGTACTTCTGAAAGC GCAACCCCTGAATCCGGTCCAG GTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAA CCTGCTACTTCTGGTTCTGAAAC CCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTA GCTCTACCCCGTCTGGTGCTACT GGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA GGTAGCTCTACCCCTTCTGGTG CTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCG CACCAGGTACCTCTACTGAACC GTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTT CTGAAACTCCAGGTAGCCCTGC TGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTC CGACTTCTACTGAGGAAGGTAC TTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAA GCGGCGCGCCAAGCACGGGAG GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCG GCTGGCTCTCCGACTTCCACCG AGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGT TCTACCAGCTCTACCGCTGAAT CTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCA CCAGGTACTTCCCCTAGCGGTG AATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCT TCCTCTCCAGGTAGCTCTACCCC GTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTA CCGGTACCGGCCCAGGTAGCGA ACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCG CTACTCCGGAATCCGGCCCAGG TAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCA GCTCTACTGCAGAATCTCCGGG CCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTA CTTCTCCTAGCGGCGAATCTTCT ACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCC AGGTAGCGAACCTGCAACCTCC GGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAG CGCACCAGGTTCTACCAGCTCT ACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGG CTCTGCATCTCCAGGTTCTACTA GCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCG TCCGAAGGCAGCGCTCCAGGTA CCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC GAACCTTCTGAAGGTAGCGCAC CAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCT AGCCCGTCTGCTTCCACTGGTA CTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA GGTAGCGAACCTGCTACCTCCG GTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCT GGTCCAGGTAGCCCTGCAGGTT CTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCA ACCGGCTCCCCAGGTTCTAGCC CGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACC AGCTCTACTGGTTCTCCAGGTA CCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACT GAACCGTCTGAGGGTAGCGCTC CAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA FVII- ANAFLEELRPGSLE 684 GCCAACGCGTTCCTGGAGGAGC 685AM1318 RECKEEQCSFEEA TACGGCCGGGCTCCCTGGAGAG REIFKDAERTKLFGGAGTGCAAGGAGGAGCAGTG WISYSDGDQCASS CTCCTTCGAGGAGGCCCGGGAG PCQNGGSCKDQLQATCTTCAAGGACGCGGAGAGGA SYICFCLPAFEGRN CGAAGCTGTTCTGGATTTCTTACCETHKDDQLICVN AGTGATGGGGACCAGTGTGCCT ENGGCEQYCSDHTCAAGTCCATGCCAGAATGGGGG GTKRSCRCHEGYS CTCCTGCAAGGACCAGCTCCAGLLADGVSCTPTVE TCCTATATCTGCTTCTGCCTCCC YPCGKIPILEKRNATGCCTTCGAGGGCCGGAACTGT SKPQGRIVGGKVC GAGACGCACAAGGATGACCAG PKGECPWQVLLLVCTGATCTGTGTGAACGAGAACG NGAQLCGGTLINTI GCGGCTGTGAGCAGTACTGCAGWVVSAAHCFDKIK TGACCACACGGGCACCAAGCGC NWRNLIAVLGEHDTCCTGTCGGTGCCACGAGGGGT LSEHDGDEQSRRV ACTCTCTGCTGGCAGACGGGGTAQVIIPSTYVPGTT GTCCTGCACACCCACAGTTGAA NHDIALLRLHQPVTATCCATGTGGAAAAATACCTA VLTDHVVPLCLPE TTCTAGAAAAAAGAAATGCCAGRTFSERTLAFVRFS CAAACCCCAAGGCCGAATTGTG LVSGWGQLLDRG GGGGGCAAGGTGTGCCCCAAAATALELMVLNVPR GGGGAGTGTCCATGGCAGGTCC LMTQDCLQQSRK TGTTGTTGGTGAATGGAGCTCAVGDSPNITEYMFC GTTGTGTGGGGGGACCCTGATC AGYSDGSKDSCKGAACACCATCTGGGTGGTCTCCG DSGGPHATHYRGT CGGCCCACTGTTTCGACAAAAT WYLTGIVSWGQGCAAGAACTGGAGGAACCTGATC CATVGHFGVYTRV GCGGTGCTGGGCGAGCACGACCSQYIEWLQKLMRS TCAGCGAGCACGACGGGGATG EPRPGVLLRAPFPG AGCAGAGCCGGCGGGTGGCGCGTSTEPSEGSAPGS AGGTCATCATCCCCAGCACGTA EPATSGSETPGSPACGTCCCGGGCACCACCAACCAC GSPTSTEEGSTSST GACATCGCGCTGCTCCGCCTGCAESPGPGTSTPESG ACCAGCCCGTGGTCCTCACTGA SASPGSTSESPSGTCCATGTGGTGCCCCTCTGCCTG APGSTSESPSGTAP CCCGAACGGACGTTCTCTGAGAGTSTPESGSASPGT GGACGCTGGCCTTCGTGCGCTT STPESGSASPGSEPCTCATTGGTCAGCGGCTGGGGC ATSGSETPGTSESA CAGCTGCTGGACCGTGGCGCCATPESGPGSPAGSPT CGGCCCTGGAGCTCATGGTCCT STEEGTSTEPSEGSCAACGTGCCCCGGCTGATGACC APGTSESATPESGP CAGGACTGCCTGCAGCAGTCACGTSTEPSEGSAPGT GGAAGGTGGGAGACTCCCCAA STEPSEGSAPGSPAATATCACGGAGTACATGTTCTG GSPTSTEEGTSTEP TGCCGGCTACTCGGATGGCAGCSEGSAPGTSTEPSE AAGGACTCCTGCAAGGGGGAC GSAPGTSESATPESAGTGGAGGCCCACATGCCACCC GPGTSESATPESGP ACTACCGGGGCACGTGGTACCTGTSTEPSEGSAPGT GACGGGCATCGTCAGCTGGGGC STEPSEGSAPGTSECAGGGCTGCGCAACCGTGGGCC SATPESGPGTSTEP ACTTTGGGGTGTACACCAGGGTSEGSAPGSEPATSG CTCCCAGTACATCGAGTGGCTG SETPGSPAGSPTSTCAAAAGCTCATGCGCTCAGAGC EEGSSTPSGATGSP CACGCCCAGGAGTCCTCCTGCGGTPGSGTASSSPGS AGCCCCATTTCCCGGTGGTACT STPSGATGSPGTSTTCTACTGAACCGTCTGAAGGCA EPSEGSAPGTSTEP GCGCACCAGGTAGCGAACCGGCSEGSAPGSEPATSG TACTTCCGGTTCTGAAACCCCA SETPGSPAGSPTSTGGTAGCCCAGCAGGTTCTCCAA EEGSPAGSPTSTEE CTTCTACTGAAGAAGGTTCTACGTSTEPSEGSAPGP CAGCTCTACCGCAGAATCTCCT EPTGPAPSGGSEPAGGTCCAGGTACCTCTACTCCGG TSGSETPGTSESAT AAAGCGGCTCTGCATCTCCAGGPESGPGSPAGSPTS TTCTACTAGCGAATCTCCTTCTG TEEGTSESATPESGGCACTGCACCAGGTTCTACTAG PGSPAGSPTSTEEG CGAATCCCCGTCTGGTACTGCTSPAGSPTSTEEGTS CCAGGTACTTCTACTCCTGAAA ESATPESGPGSPAGGCGGTTCCGCTTCTCCAGGTAC SPTSTEEGSPAGSP CTCTACTCCGGAAAGCGGTTCTTSTEEGSTSSTAES GCATCTCCAGGTAGCGAACCGG PGPGSTSESPSGTACAACCTCCGGCTCTGAAACCCC PGTSPSGESSTAPG AGGTACCTCTGAAAGCGCTACTSTSESPSGTAPGST CCTGAATCCGGCCCAGGTAGCC SESPSGTAPGTSPSCGGCAGGTTCTCCGACTTCCAC GESSTAPGTSTEPS TGAGGAAGGTACCTCTACTGAAEGSAPGTSESATPE CCTTCTGAGGGCAGCGCTCCAG SGPGTSESATPESGGTACTTCTGAAAGCGCTACCCC PGSEPATSGSETPG GGAGTCCGGTCCAGGTACTTCTTSESATPESGPGTS ACTGAACCGTCCGAAGGTAGCG ESATPESGPGTSTECACCAGGTACTTCTACCGAACC PSEGSAPGTSESAT GTCCGAGGGTAGCGCACCAGGTPESGPGTSTEPSEG AGCCCAGCAGGTTCTCCTACCT SAPGTSPSGESSTACCACCGAGGAAGGTACTTCTAC PGTSPSGESSTAPG CGAACCGTCCGAGGGTAGCGCATSPSGESSTAPGTS CCAGGTACTTCTACCGAACCTT TEPSEGSAPGSPAGCCGAGGGCAGCGCACCAGGTAC SPTSTEEGTSTEPS TTCTGAAAGCGCTACCCCTGAGEGSAPGSSPSASTG TCCGGCCCAGGTACTTCTGAAA TGPGSSTPSGATGSGCGCTACTCCTGAATCCGGTCC PGSSTPSGATGSPG AGGTACCTCTACTGAACCTTCCSSTPSGATGSPGSS GAAGGCAGCGCTCCAGGTACCT TPSGATGSPGASPGCTACCGAACCGTCCGAGGGCAG TSSTGSPGASASGA CGCACCAGGTACTTCTGAAAGCPSTGGTSPSGESST GCAACCCCTGAATCCGGTCCAG APGSTSSTAESPGPGTACTTCTACTGAACCTTCCGA GTSPSGESSTAPGT AGGTAGCGCTCCAGGTAGCGAASESATPESGPGTST CCTGCTACTTCTGGTTCTGAAAC EPSEGSAPGTSTEPCCCAGGTAGCCCGGCTGGCTCT SEGSAPGSSPSAST CCGACCTCCACCGAGGAAGGTAGTGPGSSTPSGAT GCTCTACCCCGTCTGGTGCTACT GSPGASPGTSSTGSGGTTCTCCAGGTACTCCGGGCA PGTSTPESGSASPG GCGGTACTGCTTCTTCCTCTCCATSPSGESSTAPGTS GGTAGCTCTACCCCTTCTGGTG PSGESSTAPGTSESCTACTGGCTCTCCAGGTACCTCT ATPESGPGSEPATS ACCGAACCGTCCGAGGGTAGCGGSETPGTSTEPSEG CACCAGGTACCTCTACTGAACC SAPGSTSESPSGTAGTCTGAGGGTAGCGCTCCAGGT PGSTSESPSGTAPG AGCGAACCGGCAACCTCCGGTTTSTPESGSASPGSP CTGAAACTCCAGGTAGCCCTGC AGSPTSTEEGTSESTGGCTCTCCGACTTCTACTGAG ATPESGPGTSTEPS GAAGGTAGCCCGGCTGGTTCTCEGSAPGSPAGSPTS CGACTTCTACTGAGGAAGGTAC TEEGTSESATPESGTTCTACCGAACCTTCCGAAGGT PGSEPATSGSETPG AGCGCTCCAGGTCCAGAACCAASSTPSGATGSPGAS CGGGGCCGGCCCCAAGCGGAG PGTSSTGSPGSSTPGTAGCGAACCGGCAACCTCCGG SGATGSPGSTSESP CTCTGAAACCCCAGGTACCTCTSGTAPGTSPSGESS GAAAGCGCTACTCCTGAATCCG TAPGSTSSTAESPGGCCCAGGTAGCCCGGCAGGTTC PGSSTPSGATGSPG TCCGACTTCCACTGAGGAAGGTASPGTSSTGSPGTP ACTTCTGAAAGCGCTACTCCTG GSGTASSSPGSPAGAGTCCGGCCCAGGTAGCCCGGC SPTSTEEGSPAGSP TGGCTCTCCGACTTCCACCGAGTSTEEGTSTEPSEG GAAGGTAGCCCGGCTGGCTCTC SAP CAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGAG TCCGGCCCAGGTAGCCCGGCTG GCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCA ACTTCTACTGAAGAAGGTTCTA CCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAA TCTCCGTCTGGCACCGCACCAG GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTTCTACC AGCGAATCTCCTTCTGGCACCG CTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGT ACTTCTCCTAGCGGCGAATCTT CTACCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA CCAGGTACTTCTGAAAGCGCTA CCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA TCCGGTCCAGGTAGCGAACCGG CAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT CCGGAATCTGGTCCAGGTACTT CTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACCTCTACTGAA CCTTCTGAGGGCAGCGCTCCAG GTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT ACTGAACCGTCCGAAGGTAGCG CACCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGT ACCTCTCCTAGCGGCGAATCTT CTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA CCAGGTACTTCTACCGAACCGT CCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCC ACCGAGGAAGGTACTTCTACCG AACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCA CCGGTACCGGCCCAGGTAGCTC TACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTC TGGTGCAACCGGCTCCCCAGGT AGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTAC CCCGTCTGGTGCAACCGGCTCC CCAGGTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCAGGTGC AAGCGCAAGCGGCGCGCCAAG CACGGGAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAG GTTCTACTAGCTCTACCGCTGA ATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTG CTCCAGGTACCTCTGAAAGCGC TACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGG GTAGCGCTCCAGGTACTTCTAC TGAACCGTCCGAAGGTAGCGCACCAGGTTCTAGCCCTTCTGCATC TACTGGTACTGGCCCAGGTAGC TCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGT ACTAGCTCTACCGGTTCTCCAG GTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTC CTAGCGGTGAATCTTCTACTGC TCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTA CTTCTGAAAGCGCAACCCCTGA ATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTC CAGGTACTTCTACCGAACCGTC CGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTGGTA CTGCTCCAGGTTCTACCAGCGA ATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCG GCTCCGCTTCTCCAGGTAGCCC GGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCG CAACCCCGGAGTCCGGCCCAGG TACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGCCCTG CTGGCTCTCCAACCTCCACCGA AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTA GCGAACCGGCAACCTCCGGTTC TGAAACCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCC CAGGTGCTTCTCCTGGTACTAG CTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTG GCTCTCCAGGTTCTACTAGCGA ATCCCCGTCTGGTACTGCTCCAGGTACTTCCCCTAGCGGTGAAT CTTCTACTGCTCCAGGTTCTACC AGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACCCCTTCT GGTGCAACCGGCTCTCCAGGTG CATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCGGGT AGCGGTACCGCTTCTTCCTCTCC AGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCC CGGCTGGTTCTCCGACTTCTACT GAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA FIX- YNSGKLEEFVQGN 686 TATAATTCAGGTAAATTGGAAG 687AE288 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTTGAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKDGAAGTTTTTGAAAACACTGAAA DINSYECWCPFGF GAACAACTGAATTTTGGAAGCAEGKNCELDVTCNI GTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSAGAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACATLAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSKCCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDY CTGTGAATTAGATGTAACATGTVNSTEAETILDNIT AACATTAAGAATGGCAGATGCG QSTQSFNDFTRVVAGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFCTGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA TSESATPESGPGSECATAATATTGAGGAGACAGAAC PATSGSETPGTSES ATACAGAGCAAAAGCGAAATGATPESGPGSEPATS TGATTCGAATTATTCCTCACCAC GSETPGTSESATPEAACTACAATGCAGCTATTAATA SGPGTSTEPSEGSA AGTACAACCATGACATTGCCCTPGSPAGSPTSTEEG TCTGGAACTGGACGAACCCTTA TSESATPESGPGSEGTGCTAAACAGCTACGTTACAC PATSGSETPGTSES CTATTTGCATTGCTGACAAGGAATPESGPGSPAGSP ATACACGAACATCTTCCTCAAA TSTEEGSPAGSPTSTTTGGATCTGGCTATGTAAGTG TEEGTSTEPSEGSA GCTGGGGAAGAGTCTTCCACAAPGTSESATPESGPG AGGGAGATCAGCTTTAGTTCTT TSESATPESGPGTSCAGTACCTTAGAGTTCCACTTG ESATPESGPGSEPA TTGACCGAGCCACATGTCTTCGTSGSETPGSEPATS ATCTACAAAGTTCACCATCTAT GSETPGSPAGSPTSAACAACATGTTCTGTGCTGGCT TEEGTSTEPSEGSA TCCATGAAGGAGGTAGAGATTCPGTSTEPSEGSAPG ATGTCAAGGAGATAGTGGGGG SEPATSGSETPGTSACCCCATGTTACTGAAGTGGAA ESATPESGPGTSTE GGGACCAGTTTCTTAACTGGAA PSEGSAPTTATTAGCTGGGGTGAAGAGTG TGCAATGAAAGGCAAATATGGA ATATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAAAA AACAAAGCTCACTGGGGTGGTA CCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT GCTACCTCCGGCTCTGAGACTC CAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC GAACCTGCAACCTCTGGCTCTG AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GGTACTTCTACTGAACCGTCCG AGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACC GAAGAAGGTACCTCTGAAAGCG CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGT TCTGAAACCCCAGGTACTTCTG AAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCT CCGACTTCCACCGAGGAAGGTA GCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC GAACCTTCCGAGGGCAGCGCAC CAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT TCTGAAAGCGCTACTCCTGAAT CCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA GGTAGCGAACCGGCTACTTCTG GTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA ACTCCAGGTAGCCCAGCAGGCT CTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAA GGCAGCGCACCAGGTACCTCTA CTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACC TCTGGCTCTGAAACCCCAGGTA CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCAC CA FIX- YNSGKLEEFVQGN 688TATAATTCAGGTAAATTGGAAG 689 AE864 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGAEAREVFENTERTT GAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGAESNPCLNGGSCKD GAAGTTTTTGAAAACACTGAAA DINSYECWCPFGFGAACAACTGAATTTTGGAAGCA EGKNCELDVTCNI GTATGTTGATGGAGATCAGTGTKNGRCEQFCKNSA GAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYRGCGGCAGTTGCAAGGATGACAT LAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTCFPCGRVSVSQTSK CCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDYCTGTGAATTAGATGTAACATGT VNSTEAETILDNIT AACATTAAGAATGGCAGATGCGQSTQSFNDFTRVV AGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCCQVVLNGKVDAFC TGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA SPAGSPTSTEEGTSCATAATATTGAGGAGACAGAAC ESATPESGPGTSTE ATACAGAGCAAAAGCGAAATGPSEGSAPGSPAGSP TGATTCGAATTATTCCTCACCAC TSTEEGTSTEPSEGAACTACAATGCAGCTATTAATA SAPGTSTEPSEGSA AGTACAACCATGACATTGCCCTPGTSESATPESGPG TCTGGAACTGGACGAACCCTTA SEPATSGSETPGSEGTGCTAAACAGCTACGTTACAC PATSGSETPGSPAG CTATTTGCATTGCTGACAAGGASPTSTEEGTSESAT ATACACGAACATCTTCCTCAAA PESGPGTSTEPSEGTTTGGATCTGGCTATGTAAGTG SAPGTSTEPSEGSA GCTGGGGAAGAGTCTTCCACAAPGSPAGSPTSTEEG AGGGAGATCAGCTTTAGTTCTT TSTEPSEGSAPGTSCAGTACCTTAGAGTTCCACTTG TEPSEGSAPGTSES TTGACCGAGCCACATGTCTTCGATPESGPGTSTEPS ATCTACAAAGTTCACCATCTAT EGSAPGTSESATPEAACAACATGTTCTGTGCTGGCT SGPGSEPATSGSET TCCATGAAGGAGGTAGAGATTCPGTSTEPSEGSAPG ATGTCAAGGAGATAGTGGGGG TSTEPSEGSAPGTSACCCCATGTTACTGAAGTGGAA ESATPESGPGTSES GGGACCAGTTTCTTAACTGGAAATPESGPGSPAGSP TTATTAGCTGGGGTGAAGAGTG TSTEEGTSESATPETGCAATGAAAGGCAAATATGGA SGPGSEPATSGSET ATATATACCAAGGTATCCCGGTPGTSESATPESGPG ATGTCAACTGGATTAAGGAAAA TSTEPSEGSAPGTSAACAAAGCTCACTGGGGTGGTA TEPSEGSAPGTSTE GCCCGGCTGGCTCTCCTACCTCTPSEGSAPGTSTEPS ACTGAGGAAGGTACTTCTGAAA EGSAPGTSTEPSEGGCGCTACTCCTGAGTCTGGTCC SAPGTSTEPSEGSA AGGTACCTCTACTGAACCGTCCPGSPAGSPTSTEEG GAAGGTAGCGCTCCAGGTAGCC TSTEPSEGSAPGTSCAGCAGGCTCTCCGACTTCCAC ESATPESGPGSEPA TGAGGAAGGTACTTCTACTGAATSGSETPGTSESAT CCTTCCGAAGGCAGCGCACCAG PESGPGSEPATSGSGTACCTCTACTGAACCTTCTGA ETPGTSESATPESG GGGCAGCGCTCCAGGTACTTCTPGTSTEPSEGSAPG GAAAGCGCTACCCCGGAATCTG TSESATPESGPGSPGCCCAGGTAGCGAACCGGCTAC AGSPTSTEEGSPAG TTCTGGTTCTGAAACCCCAGGTSPTSTEEGSPAGSP AGCGAACCGGCTACCTCCGGTT TSTEEGTSESATPECTGAAACTCCAGGTAGCCCGGC SGPGTSTEPSEGSA AGGCTCTCCGACCTCTACTGAGPGTSESATPESGPG GAAGGTACTTCTGAAAGCGCAA SEPATSGSETPGTSCCCCGGAGTCCGGCCCAGGTAC ESATPESGPGSEPA CTCTACCGAACCGTCTGAGGGCTSGSETPGTSESAT AGCGCACCAGGTACTTCTACCG PESGPGTSTEPSEGAACCGTCCGAGGGTAGCGCACC SAPGSPAGSPTSTE AGGTAGCCCAGCAGGTTCTCCTEGTSESATPESGPG ACCTCCACCGAGGAAGGTACTT SEPATSGSETPGTSCTACCGAACCGTCCGAGGGTAG ESATPESGPGSPAG CGCACCAGGTACCTCTACTGAASPTSTEEGSPAGSP CCTTCTGAGGGCAGCGCTCCAG TSTEEGTSTEPSEGGTACTTCTGAAAGCGCTACCCC SAPGTSESATPESG GGAGTCCGGTCCAGGTACTTCTPGTSESATPESGPG ACTGAACCGTCCGAAGGTAGCG TSESATPESGPGSECACCAGGTACTTCTGAAAGCGC PATSGSETPGSEPA AACCCCTGAATCCGGTCCAGGTTSGSETPGSPAGSP AGCGAACCGGCTACTTCTGGCT TSTEEGTSTEPSEGCTGAGACTCCAGGTACTTCTAC SAPGTSTEPSEGSA CGAACCGTCCGAAGGTAGCGCAPGSEPATSGSETPG CCAGGTACTTCTACTGAACCGT TSESATPESGPGTSCTGAAGGTAGCGCACCAGGTAC TEPSEGSAP TTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAA GCGCAACCCCGGAGTCCGGCCC AGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCT CTGAAAGCGCAACCCCTGAATC CGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG GTACCTCTGAAAGCGCTACTCC GGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCG CTCCAGGTACTTCTACTGAACC GTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAG GCAGCGCTCCAGGTACCTCTAC TGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTT CTGAAGGTAGCGCACCAGGTAC TTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAG GTTCTCCTACCTCCACCGAGGA AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT CTGAAAGCGCAACTCCTGAGTC TGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAG GTACCTCTGAAAGCGCAACCCC GGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAA CCCCAGGTACCTCTGAAAGCGC TACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGG GCAGCGCACCAGGTACTTCTGA AAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTC CGACTTCCACCGAGGAAGGTAG CCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCA GGCTCTCCGACCTCTACTGAGG AAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC TCTACCGAACCGTCTGAGGGCA GCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA GGTAGCGAACCTGCTACCTCCG GCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCT GGTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCT CCAACCTCCACCGAAGAAGGTA CCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCG GCAACCTCCGGTTCTGAAACCC CAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGC CCGGCTGGCTCTCCGACTTCCA CCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA GGTACTTCTACCGAACCTTCCG AGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCC GGCCCAGGTACTTCTGAAAGCG CTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCG GAATCTGGCCCAGGTAGCGAAC CGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC TCCGGTTCTGAAACTCCAGGTA GCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT GAACCTTCCGAAGGCAGCGCAC CAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGC GAACCTGCAACCTCTGGCTCTG AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GGTACTTCTACTGAACCGTCCG AGGGCAGCGCACCA FIX-YNSGKLEEFVQGN 690 TATAATTCAGGTAAATTGGAAG 691 AF864 LERECMEEKCSFEAGTTTGTTCAAGGGAACCTTGA EAREVFENTERTT GAGAGAATGTATGGAAGAAAA EFWKQYVDGDQCGTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKD GAAGTTTTTGAAAACACTGAAADINSYECWCPFGF GAACAACTGAATTTTGGAAGCA EGKNCELDVTCNIGTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSA GAGTCCAATCCATGTTTAAATGDNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACAT LAENQKSCEPAVPTAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSK CCTTTGGATTTGAAGGAAAGAALTRAETVFPDVDY CTGTGAATTAGATGTAACATGT VNSTEAETILDNITAACATTAAGAATGGCAGATGCG QSTQSFNDFTRVV AGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPWTGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFC TGTACTGAGGGATATCGACTTG GGSIVNEKWIVTACAGAAAACCAGAAGTCCTGTGA AHCVETGVKITVV ACCAGCAGTGCCATTTCCATGTAGEHNIEETEHTE GGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNYCTTCTAAGCTCACCCGTGCTGA NAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACTLELDEPLVLNSYV ATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFLAACCATTTTGGATAACATCACT KFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQYACTTCACTCGGGTTGTTGGTGG LRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA STSESPSGTAPGTSCATAATATTGAGGAGACAGAAC PSGESSTAPGSTSE ATACAGAGCAAAAGCGAAATGSPSGTAPGSTSESP TGATTCGAATTATTCCTCACCAC SGTAPGTSTPESGSAACTACAATGCAGCTATTAATA ASPGTSTPESGSAS AGTACAACCATGACATTGCCCTPGSTSESPSGTAPG TCTGGAACTGGACGAACCCTTA STSESPSGTAPGTSGTGCTAAACAGCTACGTTACAC PSGESSTAPGSTSE CTATTTGCATTGCTGACAAGGASPSGTAPGTSPSGE ATACACGAACATCTTCCTCAAA SSTAPGTSPSGESSTTTGGATCTGGCTATGTAAGTG TAPGSTSSTAESPG GCTGGGGAAGAGTCTTCCACAAPGTSPSGESSTAPG AGGGAGATCAGCTTTAGTTCTT TSPSGESSTAPGSTCAGTACCTTAGAGTTCCACTTG SSTAESPGPGTSTP TTGACCGAGCCACATGTCTTCGESGSASPGTSTPES ATCTACAAAGTTCACCATCTAT GSASPGSTSESPSGAACAACATGTTCTGTGCTGGCT TAPGSTSESPSGTA TCCATGAAGGAGGTAGAGATTCPGTSTPESGSASPG ATGTCAAGGAGATAGTGGGGG STSSTAESPGPGTSACCCCATGTTACTGAAGTGGAA TPESGSASPGSTSE GGGACCAGTTTCTTAACTGGAASPSGTAPGTSPSGE TTATTAGCTGGGGTGAAGAGTG SSTAPGSTSSTAESTGCAATGAAAGGCAAATATGGA PGPGTSPSGESSTA ATATATACCAAGGTATCCCGGTPGTSTPESGSASPG ATGTCAACTGGATTAAGGAAAA STSSTAESPGPGSTAACAAAGCTCACTGGGGTGGTT SSTAESPGPGSTSS CTACCAGCGAATCTCCTTCTGGTAESPGPGSTSSTA CACCGCTCCAGGTACCTCTCCT ESPGPGTSPSGESSAGCGGCGAATCTTCTACCGCTC TAPGSTSESPSGTA CAGGTTCTACTAGCGAATCTCCPGSTSESPSGTAPG TTCTGGCACTGCACCAGGTTCT TSTPESGPXXXGAACTAGCGAATCCCCGTCTGGTA SASGAPSTXXXXS CTGCTCCAGGTACTTCTACTCCTESPSGTAPGSTSES GAAAGCGGTTCCGCTTCTCCAG PSGTAPGSTSESPSGTACCTCTACTCCGGAAAGCGG GTAPGSTSESPSGT TTCTGCATCTCCAGGTTCTACCAAPGSTSESPSGTAP GCGAATCTCCTTCTGGCACCGC GSTSESPSGTAPGTTCCAGGTTCTACTAGCGAATCC STPESGSASPGTSP CCGTCTGGTACCGCACCAGGTASGESSTAPGTSPSG CTTCTCCTAGCGGCGAATCTTCT ESSTAPGSTSSTAEACCGCACCAGGTTCTACTAGCG SPGPGTSPSGESST AATCTCCGTCTGGCACTGCTCCAPGTSTPESGSASP AGGTACTTCTCCTAGCGGTGAA GSTSESPSGTAPGSTCTTCTACCGCTCCAGGTACTTC TSESPSGTAPGTSP CCCTAGCGGCGAATCTTCTACCSGESSTAPGSTSES GCTCCAGGTTCTACTAGCTCTA PSGTAPGTSTPESGCTGCAGAATCTCCGGGCCCAGG SASPGTSTPESGSA TACCTCTCCTAGCGGTGAATCTTSPGSTSESPSGTAP CTACCGCTCCAGGTACTTCTCC GTSTPESGSASPGSGAGCGGTGAATCTTCTACCGCT TSSTAESPGPGSTS CCAGGTTCTACTAGCTCTACTGESPSGTAPGSTSES CAGAATCTCCTGGCCCAGGTAC PSGTAPGTSPSGESCTCTACTCCGGAAAGCGGCTCT STAPGSTSSTAESP GCATCTCCAGGTACTTCTACCCGPGTSPSGESSTAP CTGAAAGCGGTTCTGCATCTCC GTSTPESGSASPGTAGGTTCTACTAGCGAATCTCCT SPSGESSTAPGTSP TCTGGCACTGCACCAGGTTCTASGESSTAPGTSPSG CCAGCGAATCTCCGTCTGGCAC ESSTAPGSTSSTAETGCACCAGGTACCTCTACCCCT SPGPGSTSSTAESP GAAAGCGGTTCCGCTTCTCCAGGPGTSPSGESSTAP GTTCTACCAGCTCTACCGCAGA GSSPSASTGTGPGSATCTCCTGGTCCAGGTACCTCT STPSGATGSPGSST ACTCCGGAAAGCGGCTCTGCAT PSGATGSPCTCCAGGTTCTACTAGCGAATC TCCTTCTGGCACTGCACCAGGT ACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAG CTCTACCGCTGAATCTCCGGGC CCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTAC CTCTACTCCTGAAAGCGGTTCT GCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCC AGGTTCTACTAGCTCTACTGCT GAATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCC GGGTCCAGGTTCTACCAGCTCT ACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATC TTCTACTGCACCAGGTTCTACTA GCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCT CCGTCTGGCACTGCACCAGGTA CCTCTACCCCTGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGC AAGCGGCGCGCCAAGCACGGG AXXXXXXXXTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTA CCAGCGAATCCCCGTCTGGTAC TGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAG GTTCTACTAGCGAATCTCCTTCT GGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGC TCCAGGTTCTACCAGCGAATCT CCTTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTC CGCATCTCCAGGTACTTCTCCTA GCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAA TCTTCTACTGCTCCAGGTTCTAC CAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCG GTGAATCTTCTACTGCACCAGG TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAG CGAATCTCCTTCTGGCACCGCT CCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTAC TTCTCCTAGCGGCGAATCTTCTA CCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA GGTACTTCTACCCCGGAAAGCG GCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCAT CTCCAGGTTCTACTAGCGAATC TCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCT CCGCTTCTCCAGGTTCCACTAG CTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTC CTTCTGGCACCGCTCCAGGTTCT ACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG CGGCGAATCTTCTACCGCACCA GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTC CCCGAGCGGTGAATCTTCTACT GCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG TACCTCCCCTAGCGGCGAATCT TCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCT CCAGGTACCTCCCCTAGCGGTG AATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTC CGGGTCCAGGTTCTACCAGCTC TACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAAT CTTCTACTGCACCAGGTTCTAG CCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTC TGGTGCAACTGGCTCTCCAGGT AGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA FIX- YNSGKLEEFVQGN 692 TATAATTCAGGTAAATTGGAAG 693 AG864LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTT GAGAGAATGTATGGAAGAAAAEFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKD GAAGTTTTTGAAAACACTGAAADINSYECWCPFGF GAACAACTGAATTTTGGAAGCA EGKNCELDVTCNIGTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSA GAGTCCAATCCATGTTTAAATGDNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACAT LAENQKSCEPAVPTAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSK CCTTTGGATTTGAAGGAAAGAALTRAETVFPDVDY CTGTGAATTAGATGTAACATGT VNSTEAETILDNITAACATTAAGAATGGCAGATGCG QSTQSFNDFTRVV AGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPWTGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFC TGTACTGAGGGATATCGACTTG GGSIVNEKWIVTACAGAAAACCAGAAGTCCTGTGA AHCVETGVKITVV ACCAGCAGTGCCATTTCCATGTAGEHNIEETEHTE GGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNYCTTCTAAGCTCACCCGTGCTGA NAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACTLELDEPLVLNSYV ATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFLAACCATTTTGGATAACATCACT KFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQYACTTCACTCGGGTTGTTGGTGG LRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA ASPGTSSTGSPGSSCATAATATTGAGGAGACAGAAC PSASTGTGPGSSPS ATACAGAGCAAAAGCGAAATGASTGTGPGTPGSG TGATTCGAATTATTCCTCACCAC TASSSPGSSTPSGAAACTACAATGCAGCTATTAATA TGSPGSNPSASTGT AGTACAACCATGACATTGCCCTGPGASPGTSSTGSP TCTGGAACTGGACGAACCCTTA GTPGSGTASSSPGSGTGCTAAACAGCTACGTTACAC STPSGATGSPGTPG CTATTTGCATTGCTGACAAGGASGTASSSPGASPGT ATACACGAACATCTTCCTCAAA SSTGSPGASPGTSSTTTGGATCTGGCTATGTAAGTG TGSPGTPGSGTASS GCTGGGGAAGAGTCTTCCACAASPGSSTPSGATGSP AGGGAGATCAGCTTTAGTTCTT GASPGTSSTGSPGTCAGTACCTTAGAGTTCCACTTG PGSGTASSSPGSST TTGACCGAGCCACATGTCTTCGPSGATGSPGSNPSA ATCTACAAAGTTCACCATCTAT STGTGPGSSPSASTAACAACATGTTCTGTGCTGGCT GTGPGSSTPSGAT TCCATGAAGGAGGTAGAGATTCGSPGSSTPSGATGS ATGTCAAGGAGATAGTGGGGG PGASPGTSSTGSPGACCCCATGTTACTGAAGTGGAA ASPGTSSTGSPGAS GGGACCAGTTTCTTAACTGGAAPGTSSTGSPGTPGS TTATTAGCTGGGGTGAAGAGTG GTASSSPGASPGTSTGCAATGAAAGGCAAATATGGA STGSPGASPGTSST ATATATACCAAGGTATCCCGGTGSPGASPGTSSTGS ATGTCAACTGGATTAAGGAAAA PGSSPSASTGTGPGAACAAAGCTCACTGGGGTGGTG TPGSGTASSSPGAS CTTCCCCGGGCACCAGCTCTACPGTSSTGSPGASPG TGGTTCTCCAGGTTCTAGCCCGT TSSTGSPGASPGTSCTGCTTCTACTGGTACTGGTCCA STGSPGSSTPSGAT GGTTCTAGCCCTTCTGCTTCCACGSPGSSTPSGATGS TGGTACTGGTCCAGGTACCCCG PGASPGTSSTGSPGGGTAGCGGTACCGCTTCTTCTTC TPGSGTASSSPGSS TCCAGGTAGCTCTACTCCGTCTTPSGATGSPGSSTP GGTGCTACCGGCTCTCCAGGTT SGATGSPGSSTPSGCTAACCCTTCTGCATCCACCGG ATGSPGSSPSASTG TACCGGCCCAGGTGCTTCTCCGTGPGASPGTSSTGS GGCACCAGCTCTACTGGTTCTC PGASPGTSSTGSPGCAGGTACCCCGGGCAGCGGTAC TPGSGTASSSPGAS CGCATCTTCTTCTCCAGGTAGCTPGTSSTGSPGASPG CTACTCCTTCTGGTGCAACTGGT TSSTGSPGASPGTSTCTCCAGGTACTCCTGGCAGCG STGSPGASPGTSST GTACCGCTTCTTCTTCTCCAGGTGSPGTPGSGTASSS GCTTCTCCTGGTACTAGCTCTAC PGSSTPSGATGSPGTGGTTCTCCAGGTGCTTCTCCGG TPGSGTASSSPGSS GCACTAGCTCTACTGGTTCTCCTPSGATGSPGTPGS AGGTACCCCGGGTAGCGGTACT GTASSSPGSSTPSGGCTTCTTCCTCTCCAGGTAGCTC ATGSPGSSTPSGAT TACCCCTTCTGGTGCAACCGGCGSPGSSPSASTGTG TCTCCAGGTGCTTCTCCGGGCA PGSSPSASTGTGPGCCAGCTCTACCGGTTCTCCAGG ASPGTSSTGSPGTP TACCCCGGGTAGCGGTACCGCTGSGTASSSPGSSTP TCTTCTTCTCCAGGTAGCTCTAC SGATGSPGSSPSASTCCGTCTGGTGCTACCGGCTCTC TGTGPGSSPSASTG CAGGTTCTAACCCTTCTGCATCCTGPGASPGTSSTGS ACCGGTACCGGCCCAGGTTCTA PGASPGTSSTGSPGGCCCTTCTGCTTCCACCGGTACT SSTPSGATGSPGSS GGCCCAGGTAGCTCTACCCCTTPSASTGTGPGASPG CTGGTGCTACCGGCTCCCCAGG TSSTGSPGSSPSASTAGCTCTACTCCTTCTGGTGCAA TGTGPGTPGSGTA CTGGCTCTCCAGGTGCATCTCCSSSPGSSTPSGATG GGGCACTAGCTCTACTGGTTCT SPGSSTPSGATGSPCCAGGTGCATCCCCTGGCACTA GASPGTSSTGSP GCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTG GTTCTCCAGGTACTCCTGGCAG CGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCT ACTGGTTCTCCAGGTGCTTCTCC GGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTA GCTCTACCGGTTCTCCAGGTTCT AGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAG CGGTACTGCTTCTTCCTCTCCAG GTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCC CCTGGCACTAGCTCTACTGGTT CTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTA GCTCTACTCCGTCTGGTGCAAC CGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCC AGGTGCATCCCCTGGCACCAGC TCTACCGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTC CTCTCCAGGTAGCTCTACCCCG TCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGC AACCGGCTCCCCAGGTAGCTCT ACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGC TTCCACTGGTACTGGCCCAGGT GCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCC GGGTACCAGCTCTACCGGTTCT CCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCT TCTCCGGGCACCAGCTCTACTG GTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCA GGTGCATCCCCTGGCACTAGCT CTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTT CTCCAGGTACCCCTGGTAGCGG TACTGCTTCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACC GGTTCTCCAGGTACCCCGGGTA GCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCTGGTG CTACTGGTTCTCCAGGTACTCC GGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTC TGGTGCTACTGGCTCTCCAGGT AGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCC TTCTGCATCCACCGGTACCGGT CCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGC ATCCCCGGGCACTAGCTCTACC GGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCA GGTAGCTCTACTCCTTCTGGTGC TACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGG CCCAGGTTCTAGCCCGTCTGCTT CTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTG GTTCTCCAGGTGCATCTCCTGGT ACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGC AACCGGCTCTCCAGGTTCTAGC CCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTAC CAGCTCTACCGGTTCTCCAGGT TCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGC AGCGGTACCGCATCTTCCTCTC CAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGC TCTACTCCTTCTGGTGCTACTGG CTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA FIX- YNSGKLEEFVQGN 694 TATAATTCAGGTAAATTGGAAG 695AM875 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTTGAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKDGAAGTTTTTGAAAACACTGAAA DINSYECWCPFGF GAACAACTGAATTTTGGAAGCAEGKNCELDVTCNI GTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSAGAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACATLAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSKCCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDY CTGTGAATTAGATGTAACATGTVNSTEAETILDNIT AACATTAAGAATGGCAGATGCG QSTQSFNDFTRVVAGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFCTGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA TSTEPSEGSAPGSECATAATATTGAGGAGACAGAAC PATSGSETPGSPAG ATACAGAGCAAAAGCGAAATGSPTSTEEGSTSSTA TGATTCGAATTATTCCTCACCAC ESPGPGTSTPESGSAACTACAATGCAGCTATTAATA ASPGSTSESPSGTA AGTACAACCATGACATTGCCCTPGSTSESPSGTAPG TCTGGAACTGGACGAACCCTTA TSTPESGSASPGTSGTGCTAAACAGCTACGTTACAC TPESGSASPGSEPA CTATTTGCATTGCTGACAAGGATSGSETPGTSESAT ATACACGAACATCTTCCTCAAA PESGPGSPAGSPTSTTTGGATCTGGCTATGTAAGTG TEEGTSTEPSEGSA GCTGGGGAAGAGTCTTCCACAAPGTSESATPESGPG AGGGAGATCAGCTTTAGTTCTT TSTEPSEGSAPGTSCAGTACCTTAGAGTTCCACTTG TEPSEGSAPGSPAG TTGACCGAGCCACATGTCTTCGSPTSTEEGTSTEPS ATCTACAAAGTTCACCATCTAT EGSAPGTSTEPSEGAACAACATGTTCTGTGCTGGCT SAPGTSESATPESG TCCATGAAGGAGGTAGAGATTCPGTSESATPESGPG ATGTCAAGGAGATAGTGGGGG TSTEPSEGSAPGTSACCCCATGTTACTGAAGTGGAA TEPSEGSAPGTSES GGGACCAGTTTCTTAACTGGAAATPESGPGTSTEPS TTATTAGCTGGGGTGAAGAGTG EGSAPGSEPATSGSTGCAATGAAAGGCAAATATGGA ETPGSPAGSPTSTE ATATATACCAAGGTATCCCGGTEGSSTPSGATGSPG ATGTCAACTGGATTAAGGAAAA TPGSGTASSSPGSSAACAAAGCTCACTGGGGTGGTA TPSGATGSPGTSTE CTTCTACTGAACCGTCTGAAGGPSEGSAPGTSTEPS CAGCGCACCAGGTAGCGAACCG EGSAPGSEPATSGSGCTACTTCCGGTTCTGAAACCC ETPGSPAGSPTSTE CAGGTAGCCCAGCAGGTTCTCCEGSPAGSPTSTEEG AACTTCTACTGAAGAAGGTTCT TSTEPSEGSAPGASACCAGCTCTACCGCAGAATCTC ASGAPSTGGTSES CTGGTCCAGGTACCTCTACTCCATPESGPGSPAGSP GGAAAGCGGCTCTGCATCTCCA TSTEEGSPAGSPTSGGTTCTACTAGCGAATCTCCTTC TEEGSTSSTAESPG TGGCACTGCACCAGGTTCTACTPGSTSESPSGTAPG AGCGAATCCCCGTCTGGTACTG TSPSGESSTAPGTPCTCCAGGTACTTCTACTCCTGA GSGTASSSPGSSTP AAGCGGTTCCGCTTCTCCAGGTSGATGSPGSSPSAS ACCTCTACTCCGGAAAGCGGTT TGTGPGSEPATSGSCTGCATCTCCAGGTAGCGAACC ETPGTSESATPESG GGCAACCTCCGGCTCTGAAACCPGSEPATSGSETPG CCAGGTACCTCTGAAAGCGCTA STSSTAESPGPGSTCTCCTGAATCCGGCCCAGGTAG SSTAESPGPGTSPS CCCGGCAGGTTCTCCGACTTCCGESSTAPGSEPATS ACTGAGGAAGGTACCTCTACTG GSETPGSEPATSGSAACCTTCTGAGGGCAGCGCTCC ETPGTSTEPSEGSA AGGTACTTCTGAAAGCGCTACCPGSTSSTAESPGPG CCGGAGTCCGGTCCAGGTACTT TSTPESGSASPGSTCTACTGAACCGTCCGAAGGTAG SESPSGTAPGTSTE CGCACCAGGTACTTCTACCGAAPSEGSAPGTSTEPS CCGTCCGAGGGTAGCGCACCAG EGSAPGTSTEPSEGGTAGCCCAGCAGGTTCTCCTAC SAPGSSTPSGATGS CTCCACCGAGGAAGGTACTTCTPGSSPSASTGTGPG ACCGAACCGTCCGAGGGTAGCG ASPGTSSTGSPGSECACCAGGTACTTCTACCGAACC PATSGSETPGTSES TTCCGAGGGCAGCGCACCAGGTATPESGPGSPAGSP ACTTCTGAAAGCGCTACCCCTG TSTEEGSSTPSGATAGTCCGGCCCAGGTACTTCTGA GSPGSSPSASTGTG AAGCGCTACTCCTGAATCCGGTPGASPGTSSTGSPG CCAGGTACCTCTACTGAACCTT TSESATPESGPGTSCCGAAGGCAGCGCTCCAGGTAC TEPSEGSAPGTSTE CTCTACCGAACCGTCCGAGGGC PSEGSAPAGCGCACCAGGTACTTCTGAAA GCGCAACCCCTGAATCCGGTCC AGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCG AACCTGCTACTTCTGGTTCTGA AACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAG GTAGCTCTACCCCGTCTGGTGC TACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACCCCTTCT GGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGG TAGCGCACCAGGTACCTCTACT GAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTC CGGTTCTGAAACTCCAGGTAGC CCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGT TCTCCGACTTCTACTGAGGAAG GTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGC GCAAGCGGCGCGCCAAGCACG GGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG CCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTTCTACCAGCTCTACCGCT GAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCAC CGCACCAGGTACTTCCCCTAGC GGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGC TTCTTCCTCTCCAGGTAGCTCTA CCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCAT CTACCGGTACCGGCCCAGGTAG CGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAA GCGCTACTCCGGAATCCGGCCC AGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCA CCAGCTCTACTGCAGAATCTCC GGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG GTACTTCTCCTAGCGGCGAATC TTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAA CTCCAGGTAGCGAACCTGCAAC CTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGG GCAGCGCACCAGGTTCTACCAG CTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAA GCGGCTCTGCATCTCCAGGTTC TACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCG AACCGTCCGAAGGCAGCGCTCC AGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCT CTACCGAACCTTCTGAAGGTAG CGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAG GTTCTAGCCCGTCTGCTTCCACT GGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTC TCCAGGTAGCGAACCTGCTACC TCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGA GTCTGGTCCAGGTAGCCCTGCA GGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGG TGCAACCGGCTCCCCAGGTTCT AGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGG CACCAGCTCTACTGGTTCTCCA GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTC TACTGAACCGTCTGAGGGTAGC GCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA FIX- YNSGKLEEFVQGN 696 TATAATTCAGGTAAATTGGAAG 697AG864 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTTGAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKDGAAGTTTTTGAAAACACTGAAA DINSYECWCPFGF GAACAACTGAATTTTGGAAGCAEGKNCELDVTCNI GTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSAGAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACATLAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSKCCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDY CTGTGAATTAGATGTAACATGTVNSTEAETILDNIT AACATTAAGAATGGCAGATGCG QSTQSFNDFTRVVAGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFCTGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA ASPGTSSTGSPGSSCATAATATTGAGGAGACAGAAC PSASTGTGPGSSPS ATACAGAGCAAAAGCGAAATGASTGTGPGTPGSG TGATTCGAATTATTCCTCACCAC TASSSPGSSTPSGAAACTACAATGCAGCTATTAATA TGSPGSNPSASTGT AGTACAACCATGACATTGCCCTGPGASPGTSSTGSP TCTGGAACTGGACGAACCCTTA GTPGSGTASSSPGSGTGCTAAACAGCTACGTTACAC STPSGATGSPGTPG CTATTTGCATTGCTGACAAGGASGTASSSPGASPGT ATACACGAACATCTTCCTCAAA SSTGSPGASPGTSSTTTGGATCTGGCTATGTAAGTG TGSPGTPGSGTASS GCTGGGGAAGAGTCTTCCACAASPGSSTPSGATGSP AGGGAGATCAGCTTTAGTTCTT GASPGTSSTGSPGTCAGTACCTTAGAGTTCCACTTG PGSGTASSSPGSST TTGACCGAGCCACATGTCTTCGPSGATGSPGSNPSA ATCTACAAAGTTCACCATCTAT STGTGPGSSPSASTAACAACATGTTCTGTGCTGGCT GTGPGSSTPSGAT TCCATGAAGGAGGTAGAGATTCGSPGSSTPSGATGS ATGTCAAGGAGATAGTGGGGG PGASPGTSSTGSPGACCCCATGTTACTGAAGTGGAA ASPGTSSTGSPGAS GGGACCAGTTTCTTAACTGGAAPGTSSTGSPGTPGS TTATTAGCTGGGGTGAAGAGTG GTASSSPGASPGTSTGCAATGAAAGGCAAATATGGA STGSPGASPGTSST ATATATACCAAGGTATCCCGGTGSPGASPGTSSTGS ATGTCAACTGGATTAAGGAAAA PGSSPSASTGTGPGAACAAAGCTCACTGGGGTGGTG TPGSGTASSSPGAS CTTCCCCGGGCACCAGCTCTACPGTSSTGSPGASPG TGGTTCTCCAGGTTCTAGCCCGT TSSTGSPGASPGTSCTGCTTCTACTGGTACTGGTCCA STGSPGSSTPSGAT GGTTCTAGCCCTTCTGCTTCCACGSPGSSTPSGATGS TGGTACTGGTCCAGGTACCCCG PGASPGTSSTGSPGGGTAGCGGTACCGCTTCTTCTTC TPGSGTASSSPGSS TCCAGGTAGCTCTACTCCGTCTTPSGATGSPGSSTP GGTGCTACCGGCTCTCCAGGTT SGATGSPGSSTPSGCTAACCCTTCTGCATCCACCGG ATGSPGSSPSASTG TACCGGCCCAGGTGCTTCTCCGTGPGASPGTSSTGS GGCACCAGCTCTACTGGTTCTC PGASPGTSSTGSPGCAGGTACCCCGGGCAGCGGTAC TPGSGTASSSPGAS CGCATCTTCTTCTCCAGGTAGCTPGTSSTGSPGASPG CTACTCCTTCTGGTGCAACTGGT TSSTGSPGASPGTSTCTCCAGGTACTCCTGGCAGCG STGSPGASPGTSST GTACCGCTTCTTCTTCTCCAGGTGSPGTPGSGTASSS GCTTCTCCTGGTACTAGCTCTAC PGSSTPSGATGSPGTGGTTCTCCAGGTGCTTCTCCGG TPGSGTASSSPGSS GCACTAGCTCTACTGGTTCTCCTPSGATGSPGTPGS AGGTACCCCGGGTAGCGGTACT GTASSSPGSSTPSGGCTTCTTCCTCTCCAGGTAGCTC ATGSPGSSTPSGAT TACCCCTTCTGGTGCAACCGGCGSPGSSPSASTGTG TCTCCAGGTGCTTCTCCGGGCA PGSSPSASTGTGPGCCAGCTCTACCGGTTCTCCAGG ASPGTSSTGSPGTP TACCCCGGGTAGCGGTACCGCTGSGTASSSPGSSTP TCTTCTTCTCCAGGTAGCTCTAC SGATGSPGSSPSASTCCGTCTGGTGCTACCGGCTCTC TGTGPGSSPSASTG CAGGTTCTAACCCTTCTGCATCCTGPGASPGTSSTGS ACCGGTACCGGCCCAGGTTCTA PGASPGTSSTGSPGGCCCTTCTGCTTCCACCGGTACT SSTPSGATGSPGSS GGCCCAGGTAGCTCTACCCCTTPSASTGTGPGASPG CTGGTGCTACCGGCTCCCCAGG TSSTGSPGSSPSASTAGCTCTACTCCTTCTGGTGCAA TGTGPGTPGSGTA CTGGCTCTCCAGGTGCATCTCCSSSPGSSTPSGATG GGGCACTAGCTCTACTGGTTCT SPGSSTPSGATGSPCCAGGTGCATCCCCTGGCACTA GASPGTSSTGSP GCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTG GTTCTCCAGGTACTCCTGGCAG CGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCT ACTGGTTCTCCAGGTGCTTCTCC GGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTA GCTCTACCGGTTCTCCAGGTTCT AGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAG CGGTACTGCTTCTTCCTCTCCAG GTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCC CCTGGCACTAGCTCTACTGGTT CTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTA GCTCTACTCCGTCTGGTGCAAC CGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCC AGGTGCATCCCCTGGCACCAGC TCTACCGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTC CTCTCCAGGTAGCTCTACCCCG TCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGC AACCGGCTCCCCAGGTAGCTCT ACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGC TTCCACTGGTACTGGCCCAGGT GCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCC GGGTACCAGCTCTACCGGTTCT CCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCT TCTCCGGGCACCAGCTCTACTG GTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCA GGTGCATCCCCTGGCACTAGCT CTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTT CTCCAGGTACCCCTGGTAGCGG TACTGCTTCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACC GGTTCTCCAGGTACCCCGGGTA GCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCTGGTG CTACTGGTTCTCCAGGTACTCC GGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTC TGGTGCTACTGGCTCTCCAGGT AGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCC TTCTGCATCCACCGGTACCGGT CCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGC ATCCCCGGGCACTAGCTCTACC GGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCA GGTAGCTCTACTCCTTCTGGTGC TACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGG CCCAGGTTCTAGCCCGTCTGCTT CTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTG GTTCTCCAGGTGCATCTCCTGGT ACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGC AACCGGCTCTCCAGGTTCTAGC CCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTAC CAGCTCTACCGGTTCTCCAGGT TCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGC AGCGGTACCGCATCTTCCTCTC CAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGC TCTACTCCTTCTGGTGCTACTGG CTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA FIX- YNSGKLEEFVQGN 698 TATAATTCAGGTAAATTGGAAG 699AM875 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTTGAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKDGAAGTTTTTGAAAACACTGAAA DINSYECWCPFGF GAACAACTGAATTTTGGAAGCAEGKNCELDVTCNI GTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSAGAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACATLAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSKCCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDY CTGTGAATTAGATGTAACATGTVNSTEAETILDNIT AACATTAAGAATGGCAGATGCG QSTQSFNDFTRVVAGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFCTGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA TSTEPSEGSAPGSECATAATATTGAGGAGACAGAAC PATSGSETPGSPAG ATACAGAGCAAAAGCGAAATGSPTSTEEGSTSSTA TGATTCGAATTATTCCTCACCAC ESPGPGTSTPESGSAACTACAATGCAGCTATTAATA ASPGSTSESPSGTA AGTACAACCATGACATTGCCCTPGSTSESPSGTAPG TCTGGAACTGGACGAACCCTTA TSTPESGSASPGTSGTGCTAAACAGCTACGTTACAC TPESGSASPGSEPA CTATTTGCATTGCTGACAAGGATSGSETPGTSESAT ATACACGAACATCTTCCTCAAA PESGPGSPAGSPTSTTTGGATCTGGCTATGTAAGTG TEEGTSTEPSEGSA GCTGGGGAAGAGTCTTCCACAAPGTSESATPESGPG AGGGAGATCAGCTTTAGTTCTT TSTEPSEGSAPGTSCAGTACCTTAGAGTTCCACTTG TEPSEGSAPGSPAG TTGACCGAGCCACATGTCTTCGSPTSTEEGTSTEPS ATCTACAAAGTTCACCATCTAT EGSAPGTSTEPSEGAACAACATGTTCTGTGCTGGCT SAPGTSESATPESG TCCATGAAGGAGGTAGAGATTCPGTSESATPESGPG ATGTCAAGGAGATAGTGGGGG TSTEPSEGSAPGTSACCCCATGTTACTGAAGTGGAA TEPSEGSAPGTSES GGGACCAGTTTCTTAACTGGAAATPESGPGTSTEPS TTATTAGCTGGGGTGAAGAGTG EGSAPGSEPATSGSTGCAATGAAAGGCAAATATGGA ETPGSPAGSPTSTE ATATATACCAAGGTATCCCGGTEGSSTPSGATGSPG ATGTCAACTGGATTAAGGAAAA TPGSGTASSSPGSSAACAAAGCTCACTGGGGTGGTA TPSGATGSPGTSTE CTTCTACTGAACCGTCTGAAGGPSEGSAPGTSTEPS CAGCGCACCAGGTAGCGAACCG EGSAPGSEPATSGSGCTACTTCCGGTTCTGAAACCC ETPGSPAGSPTSTE CAGGTAGCCCAGCAGGTTCTCCEGSPAGSPTSTEEG AACTTCTACTGAAGAAGGTTCT TSTEPSEGSAPGASACCAGCTCTACCGCAGAATCTC ASGAPSTGGTSES CTGGTCCAGGTACCTCTACTCCATPESGPGSPAGSP GGAAAGCGGCTCTGCATCTCCA TSTEEGSPAGSPTSGGTTCTACTAGCGAATCTCCTTC TEEGSTSSTAESPG TGGCACTGCACCAGGTTCTACTPGSTSESPSGTAPG AGCGAATCCCCGTCTGGTACTG TSPSGESSTAPGTPCTCCAGGTACTTCTACTCCTGA GSGTASSSPGSSTP AAGCGGTTCCGCTTCTCCAGGTSGATGSPGSSPSAS ACCTCTACTCCGGAAAGCGGTT TGTGPGSEPATSGSCTGCATCTCCAGGTAGCGAACC ETPGTSESATPESG GGCAACCTCCGGCTCTGAAACCPGSEPATSGSETPG CCAGGTACCTCTGAAAGCGCTA STSSTAESPGPGSTCTCCTGAATCCGGCCCAGGTAG SSTAESPGPGTSPS CCCGGCAGGTTCTCCGACTTCCGESSTAPGSEPATS ACTGAGGAAGGTACCTCTACTG GSETPGSEPATSGSAACCTTCTGAGGGCAGCGCTCC ETPGTSTEPSEGSA AGGTACTTCTGAAAGCGCTACCPGSTSSTAESPGPG CCGGAGTCCGGTCCAGGTACTT TSTPESGSASPGSTCTACTGAACCGTCCGAAGGTAG SESPSGTAPGTSTE CGCACCAGGTACTTCTACCGAAPSEGSAPGTSTEPS CCGTCCGAGGGTAGCGCACCAG EGSAPGTSTEPSEGGTAGCCCAGCAGGTTCTCCTAC SAPGSSTPSGATGS CTCCACCGAGGAAGGTACTTCTPGSSPSASTGTGPG ACCGAACCGTCCGAGGGTAGCG ASPGTSSTGSPGSECACCAGGTACTTCTACCGAACC PATSGSETPGTSES TTCCGAGGGCAGCGCACCAGGTATPESGPGSPAGSP ACTTCTGAAAGCGCTACCCCTG TSTEEGSSTPSGATAGTCCGGCCCAGGTACTTCTGA GSPGSSPSASTGTG AAGCGCTACTCCTGAATCCGGTPGASPGTSSTGSPG CCAGGTACCTCTACTGAACCTT TSESATPESGPGTSCCGAAGGCAGCGCTCCAGGTAC TEPSEGSAPGTSTE CTCTACCGAACCGTCCGAGGGC PSEGSAPAGCGCACCAGGTACTTCTGAAA GCGCAACCCCTGAATCCGGTCC AGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCG AACCTGCTACTTCTGGTTCTGA AACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAG GTAGCTCTACCCCGTCTGGTGC TACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACCCCTTCT GGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGG TAGCGCACCAGGTACCTCTACT GAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTC CGGTTCTGAAACTCCAGGTAGC CCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGT TCTCCGACTTCTACTGAGGAAG GTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGC GCAAGCGGCGCGCCAAGCACG GGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG CCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTTCTACCAGCTCTACCGCT GAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCAC CGCACCAGGTACTTCCCCTAGC GGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGC TTCTTCCTCTCCAGGTAGCTCTA CCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCAT CTACCGGTACCGGCCCAGGTAG CGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAA GCGCTACTCCGGAATCCGGCCC AGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCA CCAGCTCTACTGCAGAATCTCC GGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG GTACTTCTCCTAGCGGCGAATC TTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAA CTCCAGGTAGCGAACCTGCAAC CTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGG GCAGCGCACCAGGTTCTACCAG CTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAA GCGGCTCTGCATCTCCAGGTTC TACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCG AACCGTCCGAAGGCAGCGCTCC AGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCT CTACCGAACCTTCTGAAGGTAG CGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAG GTTCTAGCCCGTCTGCTTCCACT GGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTC TCCAGGTAGCGAACCTGCTACC TCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGA GTCTGGTCCAGGTAGCCCTGCA GGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGG TGCAACCGGCTCCCCAGGTTCT AGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGG CACCAGCTCTACTGGTTCTCCA GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTC TACTGAACCGTCTGAGGGTAGC GCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA FIX- YNSGKLEEFVQGN 700 TATAATTCAGGTAAATTGGAAG 701AM1318 LERECMEEKCSFE AGTTTGTTCAAGGGAACCTTGA EAREVFENTERTTGAGAGAATGTATGGAAGAAAA EFWKQYVDGDQC GTGTAGTTTTGAAGAAGCACGA ESNPCLNGGSCKDGAAGTTTTTGAAAACACTGAAA DINSYECWCPFGF GAACAACTGAATTTTGGAAGCAEGKNCELDVTCNI GTATGTTGATGGAGATCAGTGT KNGRCEQFCKNSAGAGTCCAATCCATGTTTAAATG DNKVVCSCTEGYR GCGGCAGTTGCAAGGATGACATLAENQKSCEPAVP TAATTCCTATGAATGTTGGTGTC FPCGRVSVSQTSKCCTTTGGATTTGAAGGAAAGAA LTRAETVFPDVDY CTGTGAATTAGATGTAACATGTVNSTEAETILDNIT AACATTAAGAATGGCAGATGCG QSTQSFNDFTRVVAGCAGTTTTGTAAAAATAGTGC GGEDAKPGQFPW TGATAACAAGGTGGTTTGCTCC QVVLNGKVDAFCTGTACTGAGGGATATCGACTTG GGSIVNEKWIVTA CAGAAAACCAGAAGTCCTGTGAAHCVETGVKITVV ACCAGCAGTGCCATTTCCATGT AGEHNIEETEHTEGGAAGAGTTTCTGTTTCACAAA QKRNVIRIIPHHNY CTTCTAAGCTCACCCGTGCTGANAAINKYNHDIAL GACTGTTTTTCCTGATGTGGACT LELDEPLVLNSYVATGTAAATTCTACTGAAGCTGA TPICIADKEYTNIFL AACCATTTTGGATAACATCACTKFGSGYVSGWGR CAAAGCACCCAATCATTTAATG VFHKGRSALVLQY ACTTCACTCGGGTTGTTGGTGGLRVPLVDRATCLR AGAAGATGCCAAACCAGGTCA STKFTIYNNMFCAATTCCCTTGGCAGGTTGTTTTGA GFHEGGRDSCQGD ATGGTAAAGTTGATGCATTCTGSGGPHVTEVEGTS TGGAGGCTCTATCGTTAATGAA FLTGIISWGEECAMAAATGGATTGTAACTGCTGCCC KGKYGIYTKVSRY ACTGTGTTGAAACTGGTGTTAAVNWIKEKTKLTGG AATTACAGTTGTCGCAGGTGAA TSTEPSEGSAPGSECATAATATTGAGGAGACAGAAC PATSGSETPGSPAG ATACAGAGCAAAAGCGAAATGSPTSTEEGSTSSTA TGATTCGAATTATTCCTCACCAC ESPGPGTSTPESGSAACTACAATGCAGCTATTAATA ASPGSTSESPSGTA AGTACAACCATGACATTGCCCTPGSTSESPSGTAPG TCTGGAACTGGACGAACCCTTA TSTPESGSASPGTSGTGCTAAACAGCTACGTTACAC TPESGSASPGSEPA CTATTTGCATTGCTGACAAGGATSGSETPGTSESAT ATACACGAACATCTTCCTCAAA PESGPGSPAGSPTSTTTGGATCTGGCTATGTAAGTG TEEGTSTEPSEGSA GCTGGGGAAGAGTCTTCCACAAPGTSESATPESGPG AGGGAGATCAGCTTTAGTTCTT TSTEPSEGSAPGTSCAGTACCTTAGAGTTCCACTTG TEPSEGSAPGSPAG TTGACCGAGCCACATGTCTTCGSPTSTEEGTSTEPS ATCTACAAAGTTCACCATCTAT EGSAPGTSTEPSEGAACAACATGTTCTGTGCTGGCT SAPGTSESATPESG TCCATGAAGGAGGTAGAGATTCPGTSESATPESGPG ATGTCAAGGAGATAGTGGGGG TSTEPSEGSAPGTSACCCCATGTTACTGAAGTGGAA TEPSEGSAPGTSES GGGACCAGTTTCTTAACTGGAAATPESGPGTSTEPS TTATTAGCTGGGGTGAAGAGTG EGSAPGSEPATSGSTGCAATGAAAGGCAAATATGGA ETPGSPAGSPTSTE ATATATACCAAGGTATCCCGGTEGSSTPSGATGSPG ATGTCAACTGGATTAAGGAAAA TPGSGTASSSPGSSAACAAAGCTCACTGGGGTGGTA TPSGATGSPGTSTE CTTCTACTGAACCGTCTGAAGGPSEGSAPGTSTEPS CAGCGCACCAGGTAGCGAACCG EGSAPGSEPATSGSGCTACTTCCGGTTCTGAAACCC ETPGSPAGSPTSTE CAGGTAGCCCAGCAGGTTCTCCEGSPAGSPTSTEEG AACTTCTACTGAAGAAGGTTCT TSTEPSEGSAPGPEACCAGCTCTACCGCAGAATCTC PTGPAPSGGSEPAT CTGGTCCAGGTACCTCTACTCCSGSETPGTSESATP GGAAAGCGGCTCTGCATCTCCA ESGPGSPAGSPTSTGGTTCTACTAGCGAATCTCCTTC EEGTSESATPESGP TGGCACTGCACCAGGTTCTACTGSPAGSPTSTEEGS AGCGAATCCCCGTCTGGTACTG PAGSPTSTEEGTSECTCCAGGTACTTCTACTCCTGA SATPESGPGSPAGS AAGCGGTTCCGCTTCTCCAGGTPTSTEEGSPAGSPT ACCTCTACTCCGGAAAGCGGTT STEEGSTSSTAESPCTGCATCTCCAGGTAGCGAACC GPGSTSESPSGTAP GGCAACCTCCGGCTCTGAAACCGTSPSGESSTAPGS CCAGGTACCTCTGAAAGCGCTA TSESPSGTAPGSTSCTCCTGAATCCGGCCCAGGTAG ESPSGTAPGTSPSG CCCGGCAGGTTCTCCGACTTCCESSTAPGTSTEPSE ACTGAGGAAGGTACCTCTACTG GSAPGTSESATPESAACCTTCTGAGGGCAGCGCTCC GPGTSESATPESGP AGGTACTTCTGAAAGCGCTACCGSEPATSGSETPGT CCGGAGTCCGGTCCAGGTACTT SESATPESGPGTSECTACTGAACCGTCCGAAGGTAG SATPESGPGTSTEP CGCACCAGGTACTTCTACCGAASEGSAPGTSESATP CCGTCCGAGGGTAGCGCACCAG ESGPGTSTEPSEGSGTAGCCCAGCAGGTTCTCCTAC APGTSPSGESSTAP CTCCACCGAGGAAGGTACTTCTGTSPSGESSTAPGT ACCGAACCGTCCGAGGGTAGCG SPSGESSTAPGTSTCACCAGGTACTTCTACCGAACC EPSEGSAPGSPAGS TTCCGAGGGCAGCGCACCAGGTPTSTEEGTSTEPSE ACTTCTGAAAGCGCTACCCCTG GSAPGSSPSASTGTAGTCCGGCCCAGGTACTTCTGA GPGSSTPSGATGSP AAGCGCTACTCCTGAATCCGGTGSSTPSGATGSPGS CCAGGTACCTCTACTGAACCTT STPSGATGSPGSSTCCGAAGGCAGCGCTCCAGGTAC PSGATGSPGASPGT CTCTACCGAACCGTCCGAGGGCSSTGSPGASASGAP AGCGCACCAGGTACTTCTGAAA STGGTSPSGESSTAGCGCAACCCCTGAATCCGGTCC PGSTSSTAESPGPG AGGTACTTCTACTGAACCTTCCTSPSGESSTAPGTS GAAGGTAGCGCTCCAGGTAGCG ESATPESGPGTSTEAACCTGCTACTTCTGGTTCTGA PSEGSAPGTSTEPS AACCCCAGGTAGCCCGGCTGGCEGSAPGSSPSASTG TCTCCGACCTCCACCGAGGAAG TGPGSSTPSGATGSGTAGCTCTACCCCGTCTGGTGC PGASPGTSSTGSPG TACTGGTTCTCCAGGTACTCCGTSTPESGSASPGTS GGCAGCGGTACTGCTTCTTCCT PSGESSTAPGTSPSCTCCAGGTAGCTCTACCCCTTCT GESSTAPGTSESAT GGTGCTACTGGCTCTCCAGGTAPESGPGSEPATSGS CCTCTACCGAACCGTCCGAGGG ETPGTSTEPSEGSATAGCGCACCAGGTACCTCTACT PGSTSESPSGTAPG GAACCGTCTGAGGGTAGCGCTCSTSESPSGTAPGTS CAGGTAGCGAACCGGCAACCTC TPESGSASPGSPAGCGGTTCTGAAACTCCAGGTAGC SPTSTEEGTSESAT CCTGCTGGCTCTCCGACTTCTACPESGPGTSTEPSEG TGAGGAAGGTAGCCCGGCTGGT SAPGSPAGSPTSTETCTCCGACTTCTACTGAGGAAG EGTSESATPESGPG GTACTTCTACCGAACCTTCCGASEPATSGSETPGSS AGGTAGCGCTCCAGGTCCAGAA TPSGATGSPGASPGCCAACGGGGCCGGCCCCAAGCG TSSTGSPGSSTPSG GAGGTAGCGAACCGGCAACCTCATGSPGSTSESPSG CGGCTCTGAAACCCCAGGTACC TAPGTSPSGESSTATCTGAAAGCGCTACTCCTGAAT PGSTSSTAESPGPG CCGGCCCAGGTAGCCCGGCAGGSSTPSGATGSPGAS TTCTCCGACTTCCACTGAGGAA PGTSSTGSPGTPGSGGTACTTCTGAAAGCGCTACTC GTASSSPGSPAGSP CTGAGTCCGGCCCAGGTAGCCCTSTEEGSPAGSPTS GGCTGGCTCTCCGACTTCCACC TEEGTSTEPSEGSAGAGGAAGGTAGCCCGGCTGGCT P CTCCAACTTCTACTGAAGAAGG TACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGG CTGGCTCTCCGACTTCCACCGA GGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTT CTACCAGCTCTACCGCTGAATC TCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCAC CAGGTACTTCCCCTAGCGGTGA ATCTTCTACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCAC CGCTCCAGGTTCTACTAGCGAA TCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATC TTCTACCGCACCAGGTACTTCT ACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC TACCCCTGAGTCCGGCCCAGGT ACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACC GGCAACCTCTGGCTCTGAAACC CCAGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTAC TTCTGAAAGCGCTACTCCGGAA TCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCC AGGTACTTCTGAAAGCGCTACC CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAG CGCACCAGGTACCTCCCCTAGC GGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATC TTCTACCGCTCCAGGTACCTCCC CTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACCGAACCG TCCGAGGGTAGCGCACCAGGTA GCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCAC CAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCT CTACTCCGTCTGGTGCAACTGG CTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGG TAGCTCTACCCCGTCTGGTGCT ACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTC CCCAGGTGCATCCCCGGGTACT AGCTCTACCGGTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAA GCACGGGAGGTACTTCTCCGAG CGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTG AATCTCCGGGCCCAGGTACTTC TCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCG CTACTCCGGAGTCTGGCCCAGG TACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA CTGAACCGTCCGAAGGTAGCGC ACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA GCTCTACTCCTTCTGGTGCTACC GGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC AGGTACTTCTACTCCGGAAAGC GGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACT GCTCCAGGTACCTCTCCTAGCG GCGAATCTTCTACTGCTCCAGGTACTTCTGAAAGCGCAACCCCT GAATCCGGTCCAGGTAGCGAAC CGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCG TCCGAAGGTAGCGCACCAGGTT CTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGC GAATCCCCTTCTGGCACCGCAC CAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGC CCGGCAGGCTCTCCGACCTCTA CTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA GGTACCTCTACCGAACCGTCTG AGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACC GAAGAAGGTACCTCTGAAAGCG CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGT TCTGAAACCCCAGGTAGCTCTA CCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACT AGCTCTACCGGTTCTCCAGGTA GCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTACTAGCG AATCCCCGTCTGGTACTGCTCC AGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTAC CAGCTCTACCGCAGAATCTCCG GGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG TGCATCCCCGGGTACCAGCTCT ACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCT CCAGGTAGCCCTGCTGGCTCTC CGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA CTGAGGAAGGTACTTCTACCGA ACCTTCCGAAGGTAGCGCTCCA*Sequence name reflects N- to C-terminus configuration of thecoagulation factor and XTEN components

TABLE 42 Exemplary CFXTEN comprising CF, cleavagesequences and XTEN sequences Amino SEQ SEQ CFXTEN Acid ID DNA NucleotideID Name* Sequence NO: Sequence NO: FVII- ANAFLEELR 702GCCAACGCGTTCCTGGAGGAGC 703 FXIa- PGSLERECK TACGGCCGGGCTCCCTGGAGAG AE288EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAER CTCCTTCGAGGAGGCCCGGGAGTKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTACCQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGGLPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCCCVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAGKRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAGCTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGTNASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAAECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAGTLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAAKNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCADGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCGVPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATCVLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATGRTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTADRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGCTQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTGEYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTTDSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCAIVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACCRVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAAPGVLLRAPF ATATCACGGAGTACATGTTCTG PGKLTRAET TGCCGGCTACTCGGATGGCAGCGGTSESATP AAGGACTCCTGCAAGGGGGAC ESGPGSEPA AGTGGAGGCCCACATGCCACCCTSGSETPGT ACTACCGGGGCACGTGGTACCT SESATPESG GACGGGCATCGTCAGCTGGGGCPGSEPATSG CAGGGCTGCGCAACCGTGGGCC SETPGTSES ACTTTGGGGTGTACACCAGGGTATPESGPGT CTCCCAGTACATCGAGTGGCTG STEPSEGSA CAAAAGCTCATGCGCTCAGAGCPGSPAGSPT CACGCCCAGGAGTCCTCCTGCG STEEGTSES AGCCCCATTTCCCGGT0GGTGGTATPESGPGS ACCTCTGAAAGCGCAACTCCTG EPATSGSET AGTCTGGCCCAGGTAGCGAACCPGTSESATP TGCTACCTCCGGCTCTGAGACT ESGPGSPAG CCAGGTACCTCTGAAAGCGCAASPTSTEEGS CCCCGGAATCTGGTCCAGGTAG PAGSPTSTE CGAACCTGCAACCTCTGGCTCTEGTSTEPSE GAAACCCCAGGTACCTCTGAAA GSAPGTSES GCGCTACTCCTGAATCTGGCCCATPESGPGT AGGTACTTCTACTGAACCGTCC SESATPESG GAGGGCAGCGCACCAGGTAGCPGTSESATP CCTGCTGGCTCTCCAACCTCCA ESGPGSEPA CCGAAGAAGGTACCTCTGAAAGTSGSETPGS CGCAACCCCTGAATCCGGCCCA EPATSGSET GGTAGCGAACCGGCAACCTCCGPGSPAGSPT GTTCTGAAACCCCAGGTACTTC STEEGTSTE TGAAAGCGCTACTCCTGAGTCCPSEGSAPGT GGCCCAGGTAGCCCGGCTGGCT STEPSEGSA CTCCGACTTCCACCGAGGAAGGPGSEPATSG TAGCCCGGCTGGCTCTCCAACT SETPGTSES TCTACTGAAGAAGGTACTTCTAATPESGPGT CCGAACCTTCCGAGGGCAGCGC STEPSEGSA ACCAGGTACTTCTGAAAGCGCT PACCCCTGAGTCCGGCCCAGGTA CTTCTGAAAGCGCTACTCCTGA ATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCC CAGGTAGCGAACCGGCTACTTC TGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTG AAACTCCAGGTAGCCCAGCAGG CTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG AAGGCAGCGCACCAGGTACCTC TACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGG TACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGC ACCA FVII- ANAFLEELR 704GCCAACGCGTTCCTGGAGGAGC 705 FXIa- PGSLERECK TACGGCCGGGCTCCCTGGAGAG AE864EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAER CTCCTTCGAGGAGGCCCGGGAGTKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTACCQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGGLPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCCCVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAGKRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAGCTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGTNASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAAECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAGTLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAAKNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCADGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCGVPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATCVLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATGRTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTADRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGCTQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTGEYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTTDSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCAIVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACCRVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAAPGVLLRAPF ATATCACGGAGTACATGTTCTG PGKLTRAET TGCCGGCTACTCGGATGGCAGCGGSPAGSPT AAGGACTCCTGCAAGGGGGAC STEEGTSES AGTGGAGGCCCACATGCCACCCATPESGPGT ACTACCGGGGCACGTGGTACCT STEPSEGSA GACGGGCATCGTCAGCTGGGGCPGSPAGSPT CAGGGCTGCGCAACCGTGGGCC STEEGTSTE ACTTTGGGGTGTACACCAGGGTPSEGSAPGT CTCCCAGTACATCGAGTGGCTG STEPSEGSA CAAAAGCTCATGCGCTCAGAGCPGTSESATP CACGCCCAGGAGTCCTCCTGCG ESGPGSEPA AGCCCCATTTCCCGGT0GGTGGTTSGSETPGS AGCCCGGCTGGCTCTCCTACCT EPATSGSET CTACTGAGGAAGGTACTTCTGAPGSPAGSPT AAGCGCTACTCCTGAGTCTGGT STEEGTSES CCAGGTACCTCTACTGAACCGTATPESGPGT CCGAAGGTAGCGCTCCAGGTAG STEPSEGSA CCCAGCAGGCTCTCCGACTTCCPGTSTEPSE ACTGAGGAAGGTACTTCTACTG GSAPGSPAG AACCTTCCGAAGGCAGCGCACCSPTSTEEGT AGGTACCTCTACTGAACCTTCT STEPSEGSA GAGGGCAGCGCTCCAGGTACTTPGTSTEPSE CTGAAAGCGCTACCCCGGAATC GSAPGTSES TGGCCCAGGTAGCGAACCGGCTATPESGPGT ACTTCTGGTTCTGAAACCCCAG STEPSEGSA GTAGCGAACCGGCTACCTCCGGPGTSESATP TTCTGAAACTCCAGGTAGCCCG ESGPGSEPA GCAGGCTCTCCGACCTCTACTGTSGSETPGT AGGAAGGTACTTCTGAAAGCGC STEPSEGSA AACCCCGGAGTCCGGCCCAGGTPGTSTEPSE ACCTCTACCGAACCGTCTGAGG GSAPGTSES GCAGCGCACCAGGTACTTCTACATPESGPGT CGAACCGTCCGAGGGTAGCGCA SESATPESG CCAGGTAGCCCAGCAGGTTCTCPGSPAGSPT CTACCTCCACCGAGGAAGGTAC STEEGTSES TTCTACCGAACCGTCCGAGGGTATPESGPGS AGCGCACCAGGTACCTCTACTG EPATSGSET AACCTTCTGAGGGCAGCGCTCCPGTSESATP AGGTACTTCTGAAAGCGCTACC ESGPGTSTE CCGGAGTCCGGTCCAGGTACTTPSEGSAPGT CTACTGAACCGTCCGAAGGTAG STEPSEGSA CGCACCAGGTACTTCTGAAAGCPGTSTEPSE GCAACCCCTGAATCCGGTCCAG GSAPGTSTE GTAGCGAACCGGCTACTTCTGGPSEGSAPGT CTCTGAGACTCCAGGTACTTCT STEPSEGSA ACCGAACCGTCCGAAGGTAGCGPGTSTEPSE CACCAGGTACTTCTACTGAACC GSAPGSPAG GTCTGAAGGTAGCGCACCAGGTSPTSTEEGT ACTTCTGAAAGCGCAACCCCGG STEPSEGSA AATCCGGCCCAGGTACCTCTGAPGTSESATP AAGCGCAACCCCGGAGTCCGGC ESGPGSEPA CCAGGTAGCCCTGCTGGCTCTCTSGSETPGT CAACCTCCACCGAAGAAGGTAC SESATPESG CTCTGAAAGCGCAACCCCTGAAPGSEPATSG TCCGGCCCAGGTAGCGAACCGG SETPGTSES CAACCTCCGGTTCTGAAACCCCATPESGPGT AGGTACCTCTGAAAGCGCTACT STEPSEGSA CCGGAGTCTGGCCCAGGTACCTPGTSESATP CTACTGAACCGTCTGAGGGTAG ESGPGSPAG CGCTCCAGGTACTTCTACTGAASPTSTEEGS CCGTCCGAAGGTAGCGCACCAG PAGSPTSTE GTACTTCTACCGAACCGTCCGAEGSPAGSPT AGGCAGCGCTCCAGGTACCTCT STEEGTSES ACTGAACCTTCCGAGGGCAGCGATPESGPGT CTCCAGGTACCTCTACCGAACC STEPSEGSA TTCTGAAGGTAGCGCACCAGGTPGTSESATP ACTTCTACCGAACCGTCCGAGG ESGPGSEPA GTAGCGCACCAGGTAGCCCAGCTSGSETPGT AGGTTCTCCTACCTCCACCGAG SESATPESG GAAGGTACTTCTACCGAACCGTPGSEPATSG CCGAGGGTAGCGCACCAGGTAC SETPGTSES CTCTGAAAGCGCAACTCCTGAGATPESGPGT TCTGGCCCAGGTAGCGAACCTG STEPSEGSA CTACCTCCGGCTCTGAGACTCCPGSPAGSPT AGGTACCTCTGAAAGCGCAACC STEEGTSES CCGGAATCTGGTCCAGGTAGCGATPESGPGS AACCTGCAACCTCTGGCTCTGA EPATSGSET AACCCCAGGTACCTCTGAAAGCPGTSESATP GCTACTCCTGAATCTGGCCCAG ESGPGSPAG GTACTTCTACTGAACCGTCCGASPTSTEEGS GGGCAGCGCACCAGGTACTTCT PAGSPTSTE GAAAGCGCTACTCCTGAGTCCGEGTSTEPSE GCCCAGGTAGCCCGGCTGGCTC GSAPGTSES TCCGACTTCCACCGAGGAAGGTATPESGPGT AGCCCGGCTGGCTCTCCAACTT SESATPESG CTACTGAAGAAGGTAGCCCGGCPGTSESATP AGGCTCTCCGACCTCTACTGAG ESGPGSEPA GAAGGTACTTCTGAAAGCGCAATSGSETPGS CCCCGGAGTCCGGCCCAGGTAC EPATSGSET CTCTACCGAACCGTCTGAGGGCPGSPAGSPT AGCGCACCAGGTACCTCTGAAA STEEGTSTE GCGCAACTCCTGAGTCTGGCCCPSEGSAPGT AGGTAGCGAACCTGCTACCTCC STEPSEGSA GGCTCTGAGACTCCAGGTACCTPGSEPATSG CTGAAAGCGCAACCCCGGAATC SETPGTSES TGGTCCAGGTAGCGAACCTGCAATPESGPGT ACCTCTGGCTCTGAAACCCCAG STEPSEGSA GTACCTCTGAAAGCGCTACTCC PTGAATCTGGCCCAGGTACTTCT ACTGAACCGTCCGAGGGCAGCG CACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGT ACCTCTGAAAGCGCAACCCCTG AATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTA CTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTG GCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTT CTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAG GTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAA CCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTT CCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTT CTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAA GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA FIX- YNSGKLEEF 706TATAATTCAGGTAAATTGGAAG 707 FXIa- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA AE288MEEKCSFEE GAGAGAATGTATGGAAGAAAA AREVFENTE GTGTAGTTTTGAAGAAGCACGARTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCAPCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATGCPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTCKNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGTCSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGCAVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTGRAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGTTILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGAVVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGANGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATGVTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCAEHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTGIIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCCALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAACIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATGVSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATAYLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTATIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGACQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTGLTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTTIYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCGTGKLTRAET ATCTACAAAGTTCACCATCTAT GGTSESATP AACAACATGTTCTGTGCTGGCTESGPGSEPA TCCATGAAGGAGGTAGAGATTC TSGSETPGT ATGTCAAGGAGATAGTGGGGGSESATPESG ACCCCATGTTACTGAAGTGGAA PGSEPATSG GGGACCAGTTTCTTAACTGGAASETPGTSES TTATTAGCTGGGGTGAAGAGTG ATPESGPGT TGCAATGAAAGGCAAATATGGASTEPSEGSA ATATATACCAAGGTATCCCGGT PGSPAGSPT ATGTCAACTGGATTAAGGAAAASTEEGTSES AACAAAGCTCACTGGGGT0GGT ATPESGPGS GGTACCTCTGAAAGCGCAACTCEPATSGSET CTGAGTCTGGCCCAGGTAGCGA PGTSESATP ACCTGCTACCTCCGGCTCTGAGESGPGSPAG ACTCCAGGTACCTCTGAAAGCG SPTSTEEGS CAACCCCGGAATCTGGTCCAGGPAGSPTSTE TAGCGAACCTGCAACCTCTGGC EGTSTEPSE TCTGAAACCCCAGGTACCTCTGGSAPGTSES AAAGCGCTACTCCTGAATCTGG ATPESGPGT CCCAGGTACTTCTACTGAACCGSESATPESG TCCGAGGGCAGCGCACCAGGTA PGTSESATP GCCCTGCTGGCTCTCCAACCTCESGPGSEPA CACCGAAGAAGGTACCTCTGAA TSGSETPGS AGCGCAACCCCTGAATCCGGCCEPATSGSET CAGGTAGCGAACCGGCAACCTC PGSPAGSPT CGGTTCTGAAACCCCAGGTACTSTEEGTSTE TCTGAAAGCGCTACTCCTGAGT PSEGSAPGT CCGGCCCAGGTAGCCCGGCTGGSTEPSEGSA CTCTCCGACTTCCACCGAGGAA PGSEPATSG GGTAGCCCGGCTGGCTCTCCAASETPGTSES CTTCTACTGAAGAAGGTACTTC ATPESGPGT TACCGAACCTTCCGAGGGCAGCSTEPSEGSA GCACCAGGTACTTCTGAAAGCG P CTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT GAATCCGGTCCAGGTACTTCTG AAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT TCTGGTTCTGAAACCCCAGGTA GCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA GGCTCTCCGACTTCCACTGAGG AAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACC TCTACTGAACCTTCTGAGGGCA GCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA GGTACCTCTGAAAGCGCTACTC CTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FIX- YNSGKLEEF 708 TATAATTCAGGTAAATTGGAAG709 FXIa- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA AE864 MEEKCSFEEGAGAGAATGTATGGAAGAAAA AREVFENTE GTGTAGTTTTGAAGAAGCACGA RTTEFWKQYGAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCA PCLNGGSCKGTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATG CPFGFEGKNGCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTC KNGRCEQFCCCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGT CSCTEGYRLAACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGC AVPFPCGRVTGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTG RAETVFPDVCAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGT TILDNITQSGGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGA VVGGEDAKPGACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGA NGKVDAFCGAACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATG VTAAHCVETACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCA EHNIEETEHATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTG IIPHHNYNATGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCC ALLELDEPLACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAA CIADKEYTNCATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATG VSGWGRVFHTGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATA YLRVPLVDRAGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTA TIYNNMFCAGTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGA CQGDSGGPHATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTG LTGIISWGEGCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTT IYTKVSRYVCAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCG TGKLTRAETATCTACAAAGTTCACCATCTAT GGSPAGSPT AACAACATGTTCTGTGCTGGCT STEEGTSESTCCATGAAGGAGGTAGAGATTC ATPESGPGT ATGTCAAGGAGATAGTGGGGG STEPSEGSAACCCCATGTTACTGAAGTGGAA PGSPAGSPT GGGACCAGTTTCTTAACTGGAA STEEGTSTETTATTAGCTGGGGTGAAGAGTG PSEGSAPGT TGCAATGAAAGGCAAATATGGA STEPSEGSAATATATACCAAGGTATCCCGGT PGTSESATP ATGTCAACTGGATTAAGGAAAA ESGPGSEPAAACAAAGCTCACTGGGGT0GGT TSGSETPGS GGTAGCCCGGCTGGCTCTCCTA EPATSGSETCCTCTACTGAGGAAGGTACTTC PGSPAGSPT TGAAAGCGCTACTCCTGAGTCT STEEGTSESGGTCCAGGTACCTCTACTGAAC ATPESGPGT CGTCCGAAGGTAGCGCTCCAGG STEPSEGSATAGCCCAGCAGGCTCTCCGACT PGTSTEPSE TCCACTGAGGAAGGTACTTCTA GSAPGSPAGCTGAACCTTCCGAAGGCAGCGC SPTSTEEGT ACCAGGTACCTCTACTGAACCT STEPSEGSATCTGAGGGCAGCGCTCCAGGTA PGTSTEPSE CTTCTGAAAGCGCTACCCCGGA GSAPGTSESATCTGGCCCAGGTAGCGAACCG ATPESGPGT GCTACTTCTGGTTCTGAAACCC STEPSEGSACAGGTAGCGAACCGGCTACCTC PGTSESATP CGGTTCTGAAACTCCAGGTAGC ESGPGSEPACCGGCAGGCTCTCCGACCTCTA TSGSETPGT CTGAGGAAGGTACTTCTGAAAG STEPSEGSACGCAACCCCGGAGTCCGGCCCA PGTSTEPSE GGTACCTCTACCGAACCGTCTG GSAPGTSESAGGGCAGCGCACCAGGTACTTC ATPESGPGT TACCGAACCGTCCGAGGGTAGC SESATPESGGCACCAGGTAGCCCAGCAGGTT PGSPAGSPT CTCCTACCTCCACCGAGGAAGG STEEGTSESTACTTCTACCGAACCGTCCGAG ATPESGPGS GGTAGCGCACCAGGTACCTCTA EPATSGSETCTGAACCTTCTGAGGGCAGCGC PGTSESATP TCCAGGTACTTCTGAAAGCGCT ESGPGTSTEACCCCGGAGTCCGGTCCAGGTA PSEGSAPGT CTTCTACTGAACCGTCCGAAGG STEPSEGSATAGCGCACCAGGTACTTCTGAA PGTSTEPSE AGCGCAACCCCTGAATCCGGTC GSAPGTSTECAGGTAGCGAACCGGCTACTTC PSEGSAPGT TGGCTCTGAGACTCCAGGTACT STEPSEGSATCTACCGAACCGTCCGAAGGTA PGTSTEPSE GCGCACCAGGTACTTCTACTGA GSAPGSPAGACCGTCTGAAGGTAGCGCACCA SPTSTEEGT GGTACTTCTGAAAGCGCAACCC STEPSEGSACGGAATCCGGCCCAGGTACCTC PGTSESATP TGAAAGCGCAACCCCGGAGTCC ESGPGSEPAGGCCCAGGTAGCCCTGCTGGCT TSGSETPGT CTCCAACCTCCACCGAAGAAGG SESATPESGTACCTCTGAAAGCGCAACCCCT PGSEPATSG GAATCCGGCCCAGGTAGCGAAC SETPGTSESCGGCAACCTCCGGTTCTGAAAC ATPESGPGT CCCAGGTACCTCTGAAAGCGCT STEPSEGSAACTCCGGAGTCTGGCCCAGGTA PGTSESATP CCTCTACTGAACCGTCTGAGGG ESGPGSPAGTAGCGCTCCAGGTACTTCTACT SPTSTEEGS GAACCGTCCGAAGGTAGCGCAC PAGSPTSTECAGGTACTTCTACCGAACCGTC EGSPAGSPT CGAAGGCAGCGCTCCAGGTACC STEEGTSESTCTACTGAACCTTCCGAGGGCA ATPESGPGT GCGCTCCAGGTACCTCTACCGA STEPSEGSAACCTTCTGAAGGTAGCGCACCA PGTSESATP GGTACTTCTACCGAACCGTCCG ESGPGSEPAAGGGTAGCGCACCAGGTAGCCC TSGSETPGT AGCAGGTTCTCCTACCTCCACC SESATPESGGAGGAAGGTACTTCTACCGAAC PGSEPATSG CGTCCGAGGGTAGCGCACCAGG SETPGTSESTACCTCTGAAAGCGCAACTCCT ATPESGPGT GAGTCTGGCCCAGGTAGCGAAC STEPSEGSACTGCTACCTCCGGCTCTGAGAC PGSPAGSPT TCCAGGTACCTCTGAAAGCGCA STEEGTSESACCCCGGAATCTGGTCCAGGTA ATPESGPGS GCGAACCTGCAACCTCTGGCTC EPATSGSETTGAAACCCCAGGTACCTCTGAA PGTSESATP AGCGCTACTCCTGAATCTGGCC ESGPGSPAGCAGGTACTTCTACTGAACCGTC SPTSTEEGS CGAGGGCAGCGCACCAGGTACT PAGSPTSTETCTGAAAGCGCTACTCCTGAGT EGTSTEPSE CCGGCCCAGGTAGCCCGGCTGG GSAPGTSESCTCTCCGACTTCCACCGAGGAA ATPESGPGT GGTAGCCCGGCTGGCTCTCCAA SESATPESGCTTCTACTGAAGAAGGTAGCCC PGTSESATP GGCAGGCTCTCCGACCTCTACT ESGPGSEPAGAGGAAGGTACTTCTGAAAGCG TSGSETPGS CAACCCCGGAGTCCGGCCCAGG EPATSGSETTACCTCTACCGAACCGTCTGAG PGSPAGSPT GGCAGCGCACCAGGTACCTCTG STEEGTSTEAAAGCGCAACTCCTGAGTCTGG PSEGSAPGT CCCAGGTAGCGAACCTGCTACC STEPSEGSATCCGGCTCTGAGACTCCAGGTA PGSEPATSG CCTCTGAAAGCGCAACCCCGGA SETPGTSESATCTGGTCCAGGTAGCGAACCT TAPESGPGT GCAACCTCTGGCTCTGAAACCC STEPSEGSACAGGTACCTCTGAAAGCGCTAC P TCCTGAATCTGGCCCAGGTACT TCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGG CTCTCCAACCTCCACCGAAGAA GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGA ACCGGCAACCTCCGGTTCTGAA ACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGG TAGCCCGGCTGGCTCTCCGACT TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGA AGAAGGTACTTCTACCGAACCT TCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGA GTCCGGCCCAGGTACTTCTGAA AGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC CCCGGAATCTGGCCCAGGTAGC GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGC TACCTCCGGTTCTGAAACTCCA GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTC TACTGAACCTTCCGAAGGCAGC GCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG TAGCGAACCTGCAACCTCTGGC TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGG CCCAGGTACTTCTACTGAACCG TCCGAGGGCAGCGCACCA FVII-ANAFLEELR 710 GCCAACGCGTTCCTGGAGGAGC 711 FXIIa- PGSLERECKTACGGCCGGGCTCCCTGGAGAG AE288 EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAERCTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSPCGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFCCAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLITCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGTGAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVSGCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKRTCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKGGTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGGTTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKIGGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEHTGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTYAACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPVCAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSETCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLLAGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLMGACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNITCCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKGGGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTGCAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYTCAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPRGGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTG PGTMTRIVGTGCCGGCTACTCGGATGGCAGC GGGTSESAT AAGGACTCCTGCAAGGGGGAC PESGPGSEPAGTGGAGGCCCACATGCCACCC ATSGSETPG ACTACCGGGGCACGTGGTACCT TSESATPESGACGGGCATCGTCAGCTGGGGC GPGSEPATS CAGGGCTGCGCAACCGTGGGCC GSETPGTSEACTTTGGGGTGTACACCAGGGT SATPESGPG CTCCCAGTACATCGAGTGGCTG TSTEPSEGSCAAAAGCTCATGCGCTCAGAGC APGSPAGSP CACGCCCAGGAGTCCTCCTGCG TSTEEGTSEAGCCCCATTTCCCGGT0GGTGGT SATPESGPG ACCTCTGAAAGCGCAACTCCTG SEPATSGSEAGTCTGGCCCAGGTAGCGAACC TPGTSESAT TGCTACCTCCGGCTCTGAGACT PESGPGSPACCAGGTACCTCTGAAAGCGCAA GSPTSTEEG CCCCGGAATCTGGTCCAGGTAG SPAGSPTSTCGAACCTGCAACCTCTGGCTCT EEGTSTEPS GAAACCCCAGGTACCTCTGAAA EGSAPGTSEGCGCTACTCCTGAATCTGGCCC SATPESGPG AGGTACTTCTACTGAACCGTCC TSESATPESGAGGGCAGCGCACCAGGTAGC GPGTSESAT CCTGCTGGCTCTCCAACCTCCA PESGPGSEPCCGAAGAAGGTACCTCTGAAAG ATSGSETPG CGCAACCCCTGAATCCGGCCCA SEPATSGSEGGTAGCGAACCGGCAACCTCCG TPGSPAGSP GTTCTGAAACCCCAGGTACTTC TSTEEGTSTTGAAAGCGCTACTCCTGAGTCC EPSEGSAPG GGCCCAGGTAGCCCGGCTGGCT TSTEPSEGSCTCCGACTTCCACCGAGGAAGG APGSEPATS TAGCCCGGCTGGCTCTCCAACT GSETPGTSETCTACTGAAGAAGGTACTTCTA SATPESGPG CCGAACCTTCCGAGGGCAGCGC TSTEPSEGSACCAGGTACTTCTGAAAGCGCT AP ACCCCTGAGTCCGGCCCAGGTA CTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAA AGCGCTACCCCGGAATCTGGCC CAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGC GAACCGGCTACCTCCGGTTCTG AAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA GGTACTTCTACTGAACCTTCCG AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGC GCTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGCACCA FVII- ANAFLEELR 712 GCCAACGCGTTCCTGGAGGAGC 713 FXIIa- PGSLERECKTACGGCCGGGCTCCCTGGAGAG AE864 EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAERCTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSPCGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFCCAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLITCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGTGAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVSGCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKRTCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKGGTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGGTTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKIGGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEHTGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTYAACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPVCAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSETCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLLAGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLMGACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNITCCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKGGGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTGCAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYTCAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPRGGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTG PGTMTRIVGTGCCGGCTACTCGGATGGCAGC GGGSPAGSP AAGGACTCCTGCAAGGGGGAC TSTEEGTSEAGTGGAGGCCCACATGCCACCC SATPESGPG ACTACCGGGGCACGTGGTACCT TSTEPSEGSGACGGGCATCGTCAGCTGGGGC APGSPAGSP CAGGGCTGCGCAACCGTGGGCC TSTEEGTSTACTTTGGGGTGTACACCAGGGT EPSEGSAPG CTCCCAGTACATCGAGTGGCTG TSTEPSEGSCAAAAGCTCATGCGCTCAGAGC APGTSESAT CACGCCCAGGAGTCCTCCTGCG PESGPGSEPAGCCCCATTTCCCGGT0GGTGGT ATSGSETPG AGCCCGGCTGGCTCTCCTACCT SEPATSGSECTACTGAGGAAGGTACTTCTGA TPGSPAGSP AAGCGCTACTCCTGAGTCTGGT TSTEEGTSECCAGGTACCTCTACTGAACCGT SATPESGPG CCGAAGGTAGCGCTCCAGGTAG TSTEPSEGSCCCAGCAGGCTCTCCGACTTCC APGTSTEPS ACTGAGGAAGGTACTTCTACTG EGSAPGSPAAACCTTCCGAAGGCAGCGCACC GSPTSTEEG AGGTACCTCTACTGAACCTTCT TSTEPSEGSGAGGGCAGCGCTCCAGGTACTT APGTSTEPS CTGAAAGCGCTACCCCGGAATC EGSAPGTSETGGCCCAGGTAGCGAACCGGCT SATPESGPG ACTTCTGGTTCTGAAACCCCAG TSTEPSEGSGTAGCGAACCGGCTACCTCCGG APGTSESAT TTCTGAAACTCCAGGTAGCCCG PESGPGSEPGCAGGCTCTCCGACCTCTACTG ATSGSETPG AGGAAGGTACTTCTGAAAGCGC TSTEPSEGSAACCCCGGAGTCCGGCCCAGGT APGTSTEPS ACCTCTACCGAACCGTCTGAGG EGSAPGTSEGCAGCGCACCAGGTACTTCTAC SATPESGPG CGAACCGTCCGAGGGTAGCGCA TSESATPESCCAGGTAGCCCAGCAGGTTCTC GPGSPAGSP CTACCTCCACCGAGGAAGGTAC TSTEEGTSETTCTACCGAACCGTCCGAGGGT SATPESGPG AGCGCACCAGGTACCTCTACTG SEPATSGSEAACCTTCTGAGGGCAGCGCTCC TPGTSESAT AGGTACTTCTGAAAGCGCTACC PESGPGTSTCCGGAGTCCGGTCCAGGTACTT EPSEGSAPG CTACTGAACCGTCCGAAGGTAG TSTEPSEGSCGCACCAGGTACTTCTGAAAGC APGTSTEPS GCAACCCCTGAATCCGGTCCAG EGSAPGTSTGTAGCGAACCGGCTACTTCTGG EPSEGSAPG CTCTGAGACTCCAGGTACTTCT TSTEPSEGSACCGAACCGTCCGAAGGTAGCG APGTSTEPS CACCAGGTACTTCTACTGAACC EGSAPGSPAGTCTGAAGGTAGCGCACCAGGT GSPTSTEEG ACTTCTGAAAGCGCAACCCCGG TSTEPSEGSAATCCGGCCCAGGTACCTCTGA APGTSESAT AAGCGCAACCCCGGAGTCCGGC PESGPGSEPCCAGGTAGCCCTGCTGGCTCTC ATSGSETPG CAACCTCCACCGAAGAAGGTAC TSESATPESCTCTGAAAGCGCAACCCCTGAA GPGSEPATS TCCGGCCCAGGTAGCGAACCGG GSETPGTSECAACCTCCGGTTCTGAAACCCC SATPESGPG AGGTACCTCTGAAAGCGCTACT TSTEPSEGSCCGGAGTCTGGCCCAGGTACCT APGTSESAT CTACTGAACCGTCTGAGGGTAG PESGPGSPACGCTCCAGGTACTTCTACTGAA GSPTSTEEG CCGTCCGAAGGTAGCGCACCAG SPAGSPTSTGTACTTCTACCGAACCGTCCGA EEGSPAGSP AGGCAGCGCTCCAGGTACCTCT TSTEEGTSEACTGAACCTTCCGAGGGCAGCG SATPESGPG CTCCAGGTACCTCTACCGAACC TSTEPSEGSTTCTGAAGGTAGCGCACCAGGT APGTSESAT ACTTCTACCGAACCGTCCGAGG PESGPGSEPGTAGCGCACCAGGTAGCCCAGC ATSGSETPG AGGTTCTCCTACCTCCACCGAG TSESATPESGAAGGTACTTCTACCGAACCGT GPGSEPATS CCGAGGGTAGCGCACCAGGTAC GSETPGTSECTCTGAAAGCGCAACTCCTGAG SATPESGPG TCTGGCCCAGGTAGCGAACCTG TSTEPSEGSCTACCTCCGGCTCTGAGACTCC APGSPAGSP AGGTACCTCTGAAAGCGCAACC TSTEEGTSECCGGAATCTGGTCCAGGTAGCG SATPESGPG AACCTGCAACCTCTGGCTCTGA SEPATSGSEAACCCCAGGTACCTCTGAAAGC TPGTSESAT GCTACTCCTGAATCTGGCCCAG PESGPGSPAGTACTTCTACTGAACCGTCCGA GSPTSTEEG GGGCAGCGCACCAGGTACTTCT SPAGSPTSTGAAAGCGCTACTCCTGAGTCCG EEGTSTEPS GCCCAGGTAGCCCGGCTGGCTC EGSAPGTSETCCGACTTCCACCGAGGAAGGT SATPESGPG AGCCCGGCTGGCTCTCCAACTT TSESATPESCTACTGAAGAAGGTAGCCCGGC GPGTSESAT AGGCTCTCCGACCTCTACTGAG PESGPGSEPGAAGGTACTTCTGAAAGCGCAA ATSGSETPG CCCCGGAGTCCGGCCCAGGTAC SEPATSGSECTCTACCGAACCGTCTGAGGGC TPGSPAGSP AGCGCACCAGGTACCTCTGAAA TSTEEGTSTGCGCAACTCCTGAGTCTGGCCC EPSEGSAPG AGGTAGCGAACCTGCTACCTCC TSTEPSEGSGGCTCTGAGACTCCAGGTACCT APGSEPATS CTGAAAGCGCAACCCCGGAATC GSETPGTSETGGTCCAGGTAGCGAACCTGCA SATPESGPG ACCTCTGGCTCTGAAACCCCAG TSTEPSEGSGTACCTCTGAAAGCGCTACTCC AP TGAATCTGGCCCAGGTACTTCT ACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTC TCCAACCTCCACCGAAGAAGGT ACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG CCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTC CGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC GGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTAC CTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTAC TGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAG CGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC AGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA FIX-YNSGKLEEF 714 TATAATTCAGGTAAATTGGAAG 715 FXIIa- VQGNLERECAGTTTGTTCAAGGGAACCTTGA AE288 MEEKCSFEE GAGAGAATGTATGGAAGAAAA AREVFENTEGTGTAGTTTTGAAGAAGCACGA RTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESNGAACAACTGAATTTTGGAAGCA PCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECWGAGTCCAATCCATGTTTAAATG CPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNITAATTCCTATGAATGTTGGTGTC KNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVVCTGTGAATTAGATGTAACATGT CSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEPAGCAGTTTTGTAAAAATAGTGC AVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLTTGTACTGAGGGATATCGACTTG RAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAEACCAGCAGTGCCATTTCCATGT TILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTRCTTCTAAGCTCACCCGTGCTGA VVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVLATGTAAATTCTACTGAAGCTGA NGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWICAAAGCACCCAATCATTTAATG VTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAGAGAAGATGCCAAACCAGGTCA EHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIRATGGTAAAGTTGATGCATTCTG IIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDIAAATGGATTGTAACTGCTGCCC ALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPIAATTACAGTTGTCGCAGGTGAA CIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGYATACAGAGCAAAAGCGAAATG VSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQAACTACAATGCAGCTATTAATA YLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKFTCTGGAACTGGACGAACCCTTA TIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDSCTATTTGCATTGCTGACAAGGA CQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSFTTTGGATCTGGCTATGTAAGTG LTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYGAGGGAGATCAGCTTTAGTTCTT IYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKLTTGACCGAGCCACATGTCTTCG TGTMTRIVG ATCTACAAAGTTCACCATCTAT GGGTSESATAACAACATGTTCTGTGCTGGCT PESGPGSEP TCCATGAAGGAGGTAGAGATTC ATSGSETPGATGTCAAGGAGATAGTGGGGG TSESATPES ACCCCATGTTACTGAAGTGGAA GPGSEPATSGGGACCAGTTTCTTAACTGGAA GSETPGTSE TTATTAGCTGGGGTGAAGAGTG SATPESGPGTGCAATGAAAGGCAAATATGGA TSTEPSEGS ATATATACCAAGGTATCCCGGT APGSPAGSPATGTCAACTGGATTAAGGAAAA TSTEEGTSE AACAAAGCTCACTGGGGT0GGT SATPESGPGGGTACCTCTGAAAGCGCAACTC SEPATSGSE CTGAGTCTGGCCCAGGTAGCGA TPGTSESATACCTGCTACCTCCGGCTCTGAG PESGPGSPA ACTCCAGGTACCTCTGAAAGCG GSPTSTEEGCAACCCCGGAATCTGGTCCAGG SPAGSPTST TAGCGAACCTGCAACCTCTGGC EEGTSTEPSTCTGAAACCCCAGGTACCTCTG EGSAPGTSE AAAGCGCTACTCCTGAATCTGG SATPESGPGCCCAGGTACTTCTACTGAACCG TSESATPES TCCGAGGGCAGCGCACCAGGTA GPGTSESATGCCCTGCTGGCTCTCCAACCTC PESGPGSEP CACCGAAGAAGGTACCTCTGAA ATSGSETPGAGCGCAACCCCTGAATCCGGCC SEPATSGSE CAGGTAGCGAACCGGCAACCTC TPGSPAGSPCGGTTCTGAAACCCCAGGTACT TSTEEGTST TCTGAAAGCGCTACTCCTGAGT EPSEGSAPGCCGGCCCAGGTAGCCCGGCTGG TSTEPSEGS CTCTCCGACTTCCACCGAGGAA APGSEPATSGGTAGCCCGGCTGGCTCTCCAA GSETPGTSE CTTCTACTGAAGAAGGTACTTC SATPESGPGTACCGAACCTTCCGAGGGCAGC TSTEPSEGS GCACCAGGTACTTCTGAAAGCG APCTACCCCTGAGTCCGGCCCAGG TACTTCTGAAAGCGCTACTCCT GAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGG CCCAGGTAGCGAACCGGCTACT TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TGAAACTCCAGGTAGCCCAGCA GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTC CGAAGGCAGCGCACCAGGTACC TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGC AACCTCTGGCTCTGAAACCCCA GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC TACTGAACCGTCCGAGGGCAGC GCACCA FIX- YNSGKLEEF 716TATAATTCAGGTAAATTGGAAG 717 FXIIa- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA AE864MEEKCSFEE GAGAGAATGTATGGAAGAAAA AREVFENTE GTGTAGTTTTGAAGAAGCACGARTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCAPCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATGCPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTCKNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGTCSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGCAVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTGRAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGTTILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGAVVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGANGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATGVTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCAEHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTGIIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCCALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAACIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATGVSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATAYLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTATIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGACQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTGLTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTTIYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCGTGTMTRIVG ATCTACAAAGTTCACCATCTAT GGGSPAGSP AACAACATGTTCTGTGCTGGCTTSTEEGTSE TCCATGAAGGAGGTAGAGATTC SATPESGPG ATGTCAAGGAGATAGTGGGGGTSTEPSEGS ACCCCATGTTACTGAAGTGGAA APGSPAGSP GGGACCAGTTTCTTAACTGGAATSTEEGTST TTATTAGCTGGGGTGAAGAGTG EPSEGSAPG TGCAATGAAAGGCAAATATGGATSTEPSEGS ATATATACCAAGGTATCCCGGT APGTSESAT ATGTCAACTGGATTAAGGAAAAPESGPGSEP AACAAAGCTCACTGGGGT0GGT ATSGSETPG GGTAGCCCGGCTGGCTCTCCTASEPATSGSE CCTCTACTGAGGAAGGTACTTC TPGSPAGSP TGAAAGCGCTACTCCTGAGTCTTSTEEGTSE GGTCCAGGTACCTCTACTGAAC SATPESGPG CGTCCGAAGGTAGCGCTCCAGGTSTEPSEGS TAGCCCAGCAGGCTCTCCGACT APGTSTEPS TCCACTGAGGAAGGTACTTCTAEGSAPGSPA CTGAACCTTCCGAAGGCAGCGC GSPTSTEEG ACCAGGTACCTCTACTGAACCTTSTEPSEGS TCTGAGGGCAGCGCTCCAGGTA APGTSTEPS CTTCTGAAAGCGCTACCCCGGAEGSAPGTSE ATCTGGCCCAGGTAGCGAACCG SATPESGPG GCTACTTCTGGTTCTGAAACCCTSTEPSEGS CAGGTAGCGAACCGGCTACCTC APGTSESAT CGGTTCTGAAACTCCAGGTAGCPESGPGSEP CCGGCAGGCTCTCCGACCTCTA ATSGSETPG CTGAGGAAGGTACTTCTGAAAGTSTEPSEGS CGCAACCCCGGAGTCCGGCCCA APGTSTEPS GGTACCTCTACCGAACCGTCTGEGSAPGTSE AGGGCAGCGCACCAGGTACTTC SATPESGPG TACCGAACCGTCCGAGGGTAGCTSESATPES GCACCAGGTAGCCCAGCAGGTT GPGSPAGSP CTCCTACCTCCACCGAGGAAGGTSTEEGTSE TACTTCTACCGAACCGTCCGAG SATPESGPG GGTAGCGCACCAGGTACCTCTASEPATSGSE CTGAACCTTCTGAGGGCAGCGC TPGTSESAT TCCAGGTACTTCTGAAAGCGCTPESGPGTST ACCCCGGAGTCCGGTCCAGGTA EPSEGSAPG CTTCTACTGAACCGTCCGAAGGTSTEPSEGS TAGCGCACCAGGTACTTCTGAA APGTSTEPS AGCGCAACCCCTGAATCCGGTCEGSAPGTST CAGGTAGCGAACCGGCTACTTC EPSEGSAPG TGGCTCTGAGACTCCAGGTACTTSTEPSEGS TCTACCGAACCGTCCGAAGGTA APGTSTEPS GCGCACCAGGTACTTCTACTGAEGSAPGSPA ACCGTCTGAAGGTAGCGCACCA GSPTSTEEG GGTACTTCTGAAAGCGCAACCCTSTEPSEGS CGGAATCCGGCCCAGGTACCTC APGTSESAT TGAAAGCGCAACCCCGGAGTCCPESGPGSEP GGCCCAGGTAGCCCTGCTGGCT ATSGSETPG CTCCAACCTCCACCGAAGAAGGTSESATPES TACCTCTGAAAGCGCAACCCCT GPGSEPATS GAATCCGGCCCAGGTAGCGAACGSETPGTSE CGGCAACCTCCGGTTCTGAAAC SATPESGPG CCCAGGTACCTCTGAAAGCGCTTSTEPSEGS ACTCCGGAGTCTGGCCCAGGTA APGTSESAT CCTCTACTGAACCGTCTGAGGGPESGPGSPA TAGCGCTCCAGGTACTTCTACT GSPTSTEEG GAACCGTCCGAAGGTAGCGCACSPAGSPTST CAGGTACTTCTACCGAACCGTC EEGSPAGSP CGAAGGCAGCGCTCCAGGTACCTSTEEGTSE TCTACTGAACCTTCCGAGGGCA SATPESGPG GCGCTCCAGGTACCTCTACCGATSTEPSEGS ACCTTCTGAAGGTAGCGCACCA APGTSESAT GGTACTTCTACCGAACCGTCCGPESGPGSEP AGGGTAGCGCACCAGGTAGCCC ATSGSETPG AGCAGGTTCTCCTACCTCCACCTSESATPES GAGGAAGGTACTTCTACCGAAC GPGSEPATS CGTCCGAGGGTAGCGCACCAGGGSETPGTSE TACCTCTGAAAGCGCAACTCCT SATPESGPG GAGTCTGGCCCAGGTAGCGAACTSTEPSEGS CTGCTACCTCCGGCTCTGAGAC APGSPAGSP TCCAGGTACCTCTGAAAGCGCATSTEEGTSE ACCCCGGAATCTGGTCCAGGTA SATPESGPG GCGAACCTGCAACCTCTGGCTCSEPATSGSE TGAAACCCCAGGTACCTCTGAA TPGTSESAT AGCGCTACTCCTGAATCTGGCCPESGPGSPA CAGGTACTTCTACTGAACCGTC GSPTSTEEG CGAGGGCAGCGCACCAGGTACTSPAGSPTST TCTGAAAGCGCTACTCCTGAGT EEGTSTEPS CCGGCCCAGGTAGCCCGGCTGGEGSAPGTSE CTCTCCGACTTCCACCGAGGAA SATPESGPG GGTAGCCCGGCTGGCTCTCCAATSESATPES CTTCTACTGAAGAAGGTAGCCC GPGTSESAT GGCAGGCTCTCCGACCTCTACTPESGPGSEP GAGGAAGGTACTTCTGAAAGCG ATSGSETPG CAACCCCGGAGTCCGGCCCAGGSEPATSGSE TACCTCTACCGAACCGTCTGAG TPGSPAGSP GGCAGCGCACCAGGTACCTCTGTSTEEGTST AAAGCGCAACTCCTGAGTCTGG EPSEGSAPG CCCAGGTAGCGAACCTGCTACCTSTEPSEGS TCCGGCTCTGAGACTCCAGGTA APGSEPATS CCTCTGAAAGCGCAACCCCGGAGSETPGTSE ATCTGGTCCAGGTAGCGAACCT SATPESGPG GCAACCTCTGGCTCTGAAACCCTSTEPSEGS CAGGTACCTCTGAAAGCGCTAC AP TCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCA GCGCACCAGGTAGCCCTGCTGG CTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCC CTGAATCCGGCCCAGGTAGCGA ACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCG CTACTCCTGAGTCCGGCCCAGG TAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGG CTGGCTCTCCAACTTCTACTGA AGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTA CTTCTGAAAGCGCTACCCCTGA GTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTC CAGGTACTTCTGAAAGCGCTAC CCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG AAACCCCAGGTAGCGAACCGGC TACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGA CTTCCACTGAGGAAGGTACTTC TACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAAC CTTCTGAGGGCAGCGCTCCAGG TAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG AAAGCGCTACTCCTGAATCTGG CCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA FVII- ANAFLEELR 718 GCCAACGCGTTCCTGGAGGAGC 719 Kal-PGSLERECK TACGGCCGGGCTCCCTGGAGAG likrein- EEQCSFEEAGGAGTGCAAGGAGGAGCAGTG AE288 REIFKDAER CTCCTTCGAGGAGGCCCGGGAG TKLFWISYSATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTAC CQNGGSCKDAGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGG LPAFEGRNCCTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCC CVNENGGCETGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAG KRSCRCHEGCTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAG CTPTVEYPCTGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGT NASKPQGRIACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAA ECPWQVLLLTATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAG TLINTIWVVCAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAA KNWRNLIAVGGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCA DGDEQSRRVGTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCG VPGTTNHDICGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATC VLTDHVVPLGCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATG RTLAFVRFSAGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTA DRGATALELCGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGC TQDCLQQSRACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTG EYMFCAGYSCCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTT DSGGPHATHCTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCA IVSWGQGCACGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACC RVSQYIEWLCAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAA PGVLLRAPFATATCACGGAGTACATGTTCTG PGSPFRSTG TGCCGGCTACTCGGATGGCAGC GGGTSESATAAGGACTCCTGCAAGGGGGAC PESGPGSEP AGTGGAGGCCCACATGCCACCC ATSGSETPGACTACCGGGGCACGTGGTACCT TSESATPES GACGGGCATCGTCAGCTGGGGC GPGSEPATSCAGGGCTGCGCAACCGTGGGCC GSETPGTSE ACTTTGGGGTGTACACCAGGGT SATPESGPGCTCCCAGTACATCGAGTGGCTG TSTEPSEGS CAAAAGCTCATGCGCTCAGAGC APGSPAGSPCACGCCCAGGAGTCCTCCTGCG TSTEEGTSE AGCCCCATTTCCCGGT0GGTGGT SATPESGPGACCTCTGAAAGCGCAACTCCTG SEPATSGSE AGTCTGGCCCAGGTAGCGAACC TPGTSESATTGCTACCTCCGGCTCTGAGACT PESGPGSPA CCAGGTACCTCTGAAAGCGCAA GSPTSTEEGCCCCGGAATCTGGTCCAGGTAG SPAGSPTST CGAACCTGCAACCTCTGGCTCT EEGTSTEPSGAAACCCCAGGTACCTCTGAAA EGSAPGTSE GCGCTACTCCTGAATCTGGCCC SATPESGPGAGGTACTTCTACTGAACCGTCC TSESATPES GAGGGCAGCGCACCAGGTAGC GPGTSESATCCTGCTGGCTCTCCAACCTCCA PESGPGSEP CCGAAGAAGGTACCTCTGAAAG ATSGSETPGCGCAACCCCTGAATCCGGCCCA SEPATSGSE GGTAGCGAACCGGCAACCTCCG TPGSPAGSPGTTCTGAAACCCCAGGTACTTC TSTEEGTST TGAAAGCGCTACTCCTGAGTCC EPSEGSAPGGGCCCAGGTAGCCCGGCTGGCT TSTEPSEGS CTCCGACTTCCACCGAGGAAGG APGSEPATSTAGCCCGGCTGGCTCTCCAACT GSETPGTSE TCTACTGAAGAAGGTACTTCTA SATPESGPGCCGAACCTTCCGAGGGCAGCGC TSTEPSEGS ACCAGGTACTTCTGAAAGCGCT APACCCCTGAGTCCGGCCCAGGTA CTTCTGAAAGCGCTACTCCTGA ATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCC CAGGTAGCGAACCGGCTACTTC TGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTG AAACTCCAGGTAGCCCAGCAGG CTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG AAGGCAGCGCACCAGGTACCTC TACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGG TACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGC ACCA FVII- ANAFLEELR 720GCCAACGCGTTCCTGGAGGAGC 721 Kal- PGSLERECK TACGGCCGGGCTCCCTGGAGAGlikrein- EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG AE864 REIFKDAERCTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSPCGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFCCAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLITCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGTGAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVSGCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKRTCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKGGTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGGTTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKIGGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEHTGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTYAACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPVCAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSETCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLLAGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLMGACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNITCCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKGGGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTGCAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYTCAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPRGGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTG PGSPFRSTGTGCCGGCTACTCGGATGGCAGC GGGSPAGSP AAGGACTCCTGCAAGGGGGAC TSTEEGTSEAGTGGAGGCCCACATGCCACCC SATPESGPG ACTACCGGGGCACGTGGTACCT TSTEPSEGSGACGGGCATCGTCAGCTGGGGC APGSPAGSP CAGGGCTGCGCAACCGTGGGCC TSTEEGTSTACTTTGGGGTGTACACCAGGGT EPSEGSAPG CTCCCAGTACATCGAGTGGCTG TSTEPSEGSCAAAAGCTCATGCGCTCAGAGC APGTSESAT CACGCCCAGGAGTCCTCCTGCG PESGPGSEPAGCCCCATTTCCCGGT0GGTGGT ATSGSETPG AGCCCGGCTGGCTCTCCTACCT SEPATSGSECTACTGAGGAAGGTACTTCTGA TPGSPAGSP AAGCGCTACTCCTGAGTCTGGT TSTEEGTSECCAGGTACCTCTACTGAACCGT SATPESGPG CCGAAGGTAGCGCTCCAGGTAG TSTEPSEGSCCCAGCAGGCTCTCCGACTTCC APGTSTEPS ACTGAGGAAGGTACTTCTACTG EGSAPGSPAAACCTTCCGAAGGCAGCGCACC GSPTSTEEG AGGTACCTCTACTGAACCTTCT TSTEPSEGSGAGGGCAGCGCTCCAGGTACTT APGTSTEPS CTGAAAGCGCTACCCCGGAATC EGSAPGTSETGGCCCAGGTAGCGAACCGGCT SATPESGPG ACTTCTGGTTCTGAAACCCCAG TSTEPSEGSGTAGCGAACCGGCTACCTCCGG APGTSESAT TTCTGAAACTCCAGGTAGCCCG PESGPGSEPGCAGGCTCTCCGACCTCTACTG ATSGSETPG AGGAAGGTACTTCTGAAAGCGC TSTEPSEGSAACCCCGGAGTCCGGCCCAGGT APGTSTEPS ACCTCTACCGAACCGTCTGAGG EGSAPGTSEGCAGCGCACCAGGTACTTCTAC SATPESGPG CGAACCGTCCGAGGGTAGCGCA TSESATPESCCAGGTAGCCCAGCAGGTTCTC GPGSPAGSP CTACCTCCACCGAGGAAGGTAC TSTEEGTSETTCTACCGAACCGTCCGAGGGT SATPESGPG AGCGCACCAGGTACCTCTACTG SEPATSGSEAACCTTCTGAGGGCAGCGCTCC TPGTSESAT AGGTACTTCTGAAAGCGCTACC PESGPGTSTCCGGAGTCCGGTCCAGGTACTT EPSEGSAPG CTACTGAACCGTCCGAAGGTAG TSTEPSEGSCGCACCAGGTACTTCTGAAAGC APGTSTEPS GCAACCCCTGAATCCGGTCCAG EGSAPGTSTGTAGCGAACCGGCTACTTCTGG EPSEGSAPG CTCTGAGACTCCAGGTACTTCT TSTEPSEGSACCGAACCGTCCGAAGGTAGCG APGTSTEPS CACCAGGTACTTCTACTGAACC EGSAPGSPAGTCTGAAGGTAGCGCACCAGGT GSPTSTEEG ACTTCTGAAAGCGCAACCCCGG TSTEPSEGSAATCCGGCCCAGGTACCTCTGA APGTSESAT AAGCGCAACCCCGGAGTCCGGC PESGPGSEPCCAGGTAGCCCTGCTGGCTCTC ATSGSETPG CAACCTCCACCGAAGAAGGTAC TSESATPESCTCTGAAAGCGCAACCCCTGAA GPGSEPATS TCCGGCCCAGGTAGCGAACCGG GSETPGTSECAACCTCCGGTTCTGAAACCCC SATPESGPG AGGTACCTCTGAAAGCGCTACT TSTEPSEGSCCGGAGTCTGGCCCAGGTACCT APGTSESAT CTACTGAACCGTCTGAGGGTAG PESGPGSPACGCTCCAGGTACTTCTACTGAA GSPTSTEEG CCGTCCGAAGGTAGCGCACCAG SPAGSPTSTGTACTTCTACCGAACCGTCCGA EEGSPAGSP AGGCAGCGCTCCAGGTACCTCT TSTEEGTSEACTGAACCTTCCGAGGGCAGCG SATPESGPG CTCCAGGTACCTCTACCGAACC TSTEPSEGSTTCTGAAGGTAGCGCACCAGGT APGTSESAT ACTTCTACCGAACCGTCCGAGG PESGPGSEPGTAGCGCACCAGGTAGCCCAGC ATSGSETPG AGGTTCTCCTACCTCCACCGAG TSESATPESGAAGGTACTTCTACCGAACCGT GPGSEPATS CCGAGGGTAGCGCACCAGGTAC GSETPGTSECTCTGAAAGCGCAACTCCTGAG SATPESGPG TCTGGCCCAGGTAGCGAACCTG TSTEPSEGSCTACCTCCGGCTCTGAGACTCC APGSPAGSP AGGTACCTCTGAAAGCGCAACC TSTEEGTSECCGGAATCTGGTCCAGGTAGCG SATPESGPG AACCTGCAACCTCTGGCTCTGA SEPATSGSEAACCCCAGGTACCTCTGAAAGC TPGTSESAT GCTACTCCTGAATCTGGCCCAG PESGPGSPAGTACTTCTACTGAACCGTCCGA GSPTSTEEG GGGCAGCGCACCAGGTACTTCT SPAGSPTSTGAAAGCGCTACTCCTGAGTCCG EEGTSTEPS GCCCAGGTAGCCCGGCTGGCTC EGSAPGTSETCCGACTTCCACCGAGGAAGGT SATPESGPG AGCCCGGCTGGCTCTCCAACTT TSESATPESCTACTGAAGAAGGTAGCCCGGC GPGTSESAT AGGCTCTCCGACCTCTACTGAG PESGPGSEPGAAGGTACTTCTGAAAGCGCAA ATSGSETPG CCCCGGAGTCCGGCCCAGGTAC SEPATSGSECTCTACCGAACCGTCTGAGGGC TPGSPAGSP AGCGCACCAGGTACCTCTGAAA TSTEEGTSTGCGCAACTCCTGAGTCTGGCCC EPSEGSAPG AGGTAGCGAACCTGCTACCTCC TSTEPSEGSGGCTCTGAGACTCCAGGTACCT APGSEPATS CTGAAAGCGCAACCCCGGAATC GSETPGTSETGGTCCAGGTAGCGAACCTGCA SATPESGPG ACCTCTGGCTCTGAAACCCCAG TSTEPSEGSGTACCTCTGAAAGCGCTACTCC AP TGAATCTGGCCCAGGTACTTCT ACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTC TCCAACCTCCACCGAAGAAGGT ACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG CCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTC CGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC GGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTAC CTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTAC TGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAG CGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC AGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA FIX-YNSGKLEEF 722 TATAATTCAGGTAAATTGGAAG 723 Kal- VQGNLERECAGTTTGTTCAAGGGAACCTTGA likrein- MEEKCSFEE GAGAGAATGTATGGAAGAAAA AE288AREVFENTE GTGTAGTTTTGAAGAAGCACGA RTTEFWKQY GAAGTTTTTGAAAACACTGAAAVDGDQCESN GAACAACTGAATTTTGGAAGCA PCLNGGSCK GTATGTTGATGGAGATCAGTGTDDINSYECW GAGTCCAATCCATGTTTAAATG CPFGFEGKN GCGGCAGTTGCAAGGATGACATCELDVTCNI TAATTCCTATGAATGTTGGTGTC KNGRCEQFC CCTTTGGATTTGAAGGAAAGAAKNSADNKVV CTGTGAATTAGATGTAACATGT CSCTEGYRL AACATTAAGAATGGCAGATGCGAENQKSCEP AGCAGTTTTGTAAAAATAGTGC AVPFPCGRV TGATAACAAGGTGGTTTGCTCCSVSQTSKLT TGTACTGAGGGATATCGACTTG RAETVFPDV CAGAAAACCAGAAGTCCTGTGADYVNSTEAE ACCAGCAGTGCCATTTCCATGT TILDNITQS GGAAGAGTTTCTGTTTCACAAATQSFNDFTR CTTCTAAGCTCACCCGTGCTGA VVGGEDAKP GACTGTTTTTCCTGATGTGGACTGQFPWQVVL ATGTAAATTCTACTGAAGCTGA NGKVDAFCG AACCATTTTGGATAACATCACTGSIVNEKWI CAAAGCACCCAATCATTTAATG VTAAHCVET ACTTCACTCGGGTTGTTGGTGGGVKITVVAG AGAAGATGCCAAACCAGGTCA EHNIEETEH ATTCCCTTGGCAGGTTGTTTTGATEQKRNVIR ATGGTAAAGTTGATGCATTCTG IIPHHNYNA TGGAGGCTCTATCGTTAATGAAAINKYNHDI AAATGGATTGTAACTGCTGCCC ALLELDEPL ACTGTGTTGAAACTGGTGTTAAVLNSYVTPI AATTACAGTTGTCGCAGGTGAA CIADKEYTN CATAATATTGAGGAGACAGAACIFLKFGSGY ATACAGAGCAAAAGCGAAATG VSGWGRVFH TGATTCGAATTATTCCTCACCACKGRSALVLQ AACTACAATGCAGCTATTAATA YLRVPLVDR AGTACAACCATGACATTGCCCTATCLRSTKF TCTGGAACTGGACGAACCCTTA TIYNNMFCA GTGCTAAACAGCTACGTTACACGFHEGGRDS CTATTTGCATTGCTGACAAGGA CQGDSGGPH ATACACGAACATCTTCCTCAAAVTEVEGTSF TTTGGATCTGGCTATGTAAGTG LTGIISWGE GCTGGGGAAGAGTCTTCCACAAECAMKGKYG AGGGAGATCAGCTTTAGTTCTT IYTKVSRYV CAGTACCTTAGAGTTCCACTTGNWIKEKTKL TTGACCGAGCCACATGTCTTCG TGSPFRSTG ATCTACAAAGTTCACCATCTATGGGTSESAT AACAACATGTTCTGTGCTGGCT PESGPGSEP TCCATGAAGGAGGTAGAGATTCATSGSETPG ATGTCAAGGAGATAGTGGGGG TSESATPES ACCCCATGTTACTGAAGTGGAAGPGSEPATS GGGACCAGTTTCTTAACTGGAA GSETPGTSE TTATTAGCTGGGGTGAAGAGTGSATPESGPG TGCAATGAAAGGCAAATATGGA TSTEPSEGS ATATATACCAAGGTATCCCGGTAPGSPAGSP ATGTCAACTGGATTAAGGAAAA TSTEEGTSE AACAAAGCTCACTGGGGT0GGTSATPESGPG GGTACCTCTGAAAGCGCAACTC SEPATSGSE CTGAGTCTGGCCCAGGTAGCGATPGTSESAT ACCTGCTACCTCCGGCTCTGAG PESGPGSPA ACTCCAGGTACCTCTGAAAGCGGSPTSTEEG CAACCCCGGAATCTGGTCCAGG SPAGSPTST TAGCGAACCTGCAACCTCTGGCEEGTSTEPS TCTGAAACCCCAGGTACCTCTG EGSAPGTSE AAAGCGCTACTCCTGAATCTGGSATPESGPG CCCAGGTACTTCTACTGAACCG TSESATPES TCCGAGGGCAGCGCACCAGGTAGPGTSESAT GCCCTGCTGGCTCTCCAACCTC PESGPGSEP CACCGAAGAAGGTACCTCTGAAATSGSETPG AGCGCAACCCCTGAATCCGGCC SEPATSGSE CAGGTAGCGAACCGGCAACCTCTPGSPAGSP CGGTTCTGAAACCCCAGGTACT TSTEEGTST TCTGAAAGCGCTACTCCTGAGTEPSEGSAPG CCGGCCCAGGTAGCCCGGCTGG TSTEPSEGS CTCTCCGACTTCCACCGAGGAAAPGSEPATS GGTAGCCCGGCTGGCTCTCCAA GSETPGTSE CTTCTACTGAAGAAGGTACTTCSATPESGPG TACCGAACCTTCCGAGGGCAGC TSTEPSEGS GCACCAGGTACTTCTGAAAGCG APCTACCCCTGAGTCCGGCCCAGG TACTTCTGAAAGCGCTACTCCT GAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGG CCCAGGTAGCGAACCGGCTACT TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TGAAACTCCAGGTAGCCCAGCA GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTC CGAAGGCAGCGCACCAGGTACC TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGC AACCTCTGGCTCTGAAACCCCA GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC TACTGAACCGTCCGAGGGCAGC GCACCA FIX- YNSGKLEEF 724TATAATTCAGGTAAATTGGAAG 725 Kal- VQGNLEREC AGTTTGTTCAAGGGAACCTTGAlikrein- MEEKCSFEE GAGAGAATGTATGGAAGAAAA AE864 AREVFENTEGTGTAGTTTTGAAGAAGCACGA RTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESNGAACAACTGAATTTTGGAAGCA PCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECWGAGTCCAATCCATGTTTAAATG CPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNITAATTCCTATGAATGTTGGTGTC KNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVVCTGTGAATTAGATGTAACATGT CSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEPAGCAGTTTTGTAAAAATAGTGC AVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLTTGTACTGAGGGATATCGACTTG RAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAEACCAGCAGTGCCATTTCCATGT TILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTRCTTCTAAGCTCACCCGTGCTGA VVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVLATGTAAATTCTACTGAAGCTGA NGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWICAAAGCACCCAATCATTTAATG VTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAGAGAAGATGCCAAACCAGGTCA EHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIRATGGTAAAGTTGATGCATTCTG IIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDIAAATGGATTGTAACTGCTGCCC ALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPIAATTACAGTTGTCGCAGGTGAA CIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGYATACAGAGCAAAAGCGAAATG VSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQAACTACAATGCAGCTATTAATA YLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKFTCTGGAACTGGACGAACCCTTA TIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDSCTATTTGCATTGCTGACAAGGA CQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSFTTTGGATCTGGCTATGTAAGTG LTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYGAGGGAGATCAGCTTTAGTTCTT IYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKLTTGACCGAGCCACATGTCTTCG TGSPFRSTG ATCTACAAAGTTCACCATCTAT GGGSPAGSPAACAACATGTTCTGTGCTGGCT TSTEEGTSE TCCATGAAGGAGGTAGAGATTC SATPESGPGATGTCAAGGAGATAGTGGGGG TSTEPSEGS ACCCCATGTTACTGAAGTGGAA APGSPAGSPGGGACCAGTTTCTTAACTGGAA TSTEEGTST TTATTAGCTGGGGTGAAGAGTG EPSEGSAPGTGCAATGAAAGGCAAATATGGA TSTEPSEGS ATATATACCAAGGTATCCCGGT APGTSESATATGTCAACTGGATTAAGGAAAA PESGPGSEP AACAAAGCTCACTGGGGT0GGT ATSGSETPGGGTAGCCCGGCTGGCTCTCCTA SEPATSGSE CCTCTACTGAGGAAGGTACTTC TPGSPAGSPTGAAAGCGCTACTCCTGAGTCT TSTEEGTSE GGTCCAGGTACCTCTACTGAAC SATPESGPGCGTCCGAAGGTAGCGCTCCAGG TSTEPSEGS TAGCCCAGCAGGCTCTCCGACT APGTSTEPSTCCACTGAGGAAGGTACTTCTA EGSAPGSPA CTGAACCTTCCGAAGGCAGCGC GSPTSTEEGACCAGGTACCTCTACTGAACCT TSTEPSEGS TCTGAGGGCAGCGCTCCAGGTA APGTSTEPSCTTCTGAAAGCGCTACCCCGGA EGSAPGTSE ATCTGGCCCAGGTAGCGAACCG SATPESGPGGCTACTTCTGGTTCTGAAACCC TSTEPSEGS CAGGTAGCGAACCGGCTACCTC APGTSESATCGGTTCTGAAACTCCAGGTAGC PESGPGSEP CCGGCAGGCTCTCCGACCTCTA ATSGSETPGCTGAGGAAGGTACTTCTGAAAG TSTEPSEGS CGCAACCCCGGAGTCCGGCCCA APGTSTEPSGGTACCTCTACCGAACCGTCTG EGSAPGTSE AGGGCAGCGCACCAGGTACTTC SATPESGPGTACCGAACCGTCCGAGGGTAGC TSESATPES GCACCAGGTAGCCCAGCAGGTT GPGSPAGSPCTCCTACCTCCACCGAGGAAGG TSTEEGTSE TACTTCTACCGAACCGTCCGAG SATPESGPGGGTAGCGCACCAGGTACCTCTA SEPATSGSE CTGAACCTTCTGAGGGCAGCGC TPGTSESATTCCAGGTACTTCTGAAAGCGCT PESGPGTST ACCCCGGAGTCCGGTCCAGGTA EPSEGSAPGCTTCTACTGAACCGTCCGAAGG TSTEPSEGS TAGCGCACCAGGTACTTCTGAA APGTSTEPSAGCGCAACCCCTGAATCCGGTC EGSAPGTST CAGGTAGCGAACCGGCTACTTC EPSEGSAPGTGGCTCTGAGACTCCAGGTACT TSTEPSEGS TCTACCGAACCGTCCGAAGGTA APGTSTEPSGCGCACCAGGTACTTCTACTGA EGSAPGSPA ACCGTCTGAAGGTAGCGCACCA GSPTSTEEGGGTACTTCTGAAAGCGCAACCC TSTEPSEGS CGGAATCCGGCCCAGGTACCTC APGTSESATTGAAAGCGCAACCCCGGAGTCC PESGPGSEP GGCCCAGGTAGCCCTGCTGGCT ATSGSETPGCTCCAACCTCCACCGAAGAAGG TSESATPES TACCTCTGAAAGCGCAACCCCT GPGSEPATSGAATCCGGCCCAGGTAGCGAAC GSETPGTSE CGGCAACCTCCGGTTCTGAAAC SATPESGPGCCCAGGTACCTCTGAAAGCGCT TSTEPSEGS ACTCCGGAGTCTGGCCCAGGTA APGTSESATCCTCTACTGAACCGTCTGAGGG PESGPGSPA TAGCGCTCCAGGTACTTCTACT GSPTSTEEGGAACCGTCCGAAGGTAGCGCAC SPAGSPTST CAGGTACTTCTACCGAACCGTC EEGSPAGSPCGAAGGCAGCGCTCCAGGTACC TSTEEGTSE TCTACTGAACCTTCCGAGGGCA SATPESGPGGCGCTCCAGGTACCTCTACCGA TSTEPSEGS ACCTTCTGAAGGTAGCGCACCA APGTSESATGGTACTTCTACCGAACCGTCCG PESGPGSEP AGGGTAGCGCACCAGGTAGCCC ATSGSETPGAGCAGGTTCTCCTACCTCCACC TSESATPES GAGGAAGGTACTTCTACCGAAC GPGSEPATSCGTCCGAGGGTAGCGCACCAGG GSETPGTSE TACCTCTGAAAGCGCAACTCCT SATPESGPGGAGTCTGGCCCAGGTAGCGAAC TSTEPSEGS CTGCTACCTCCGGCTCTGAGAC APGSPAGSPTCCAGGTACCTCTGAAAGCGCA TSTEEGTSE ACCCCGGAATCTGGTCCAGGTA SATPESGPGGCGAACCTGCAACCTCTGGCTC SEPATSGSE TGAAACCCCAGGTACCTCTGAA TPGTSESATAGCGCTACTCCTGAATCTGGCC PESGPGSPA CAGGTACTTCTACTGAACCGTC GSPTSTEEGCGAGGGCAGCGCACCAGGTACT SPAGSPTST TCTGAAAGCGCTACTCCTGAGT EEGTSTEPSCCGGCCCAGGTAGCCCGGCTGG EGSAPGTSE CTCTCCGACTTCCACCGAGGAA SATPESGPGGGTAGCCCGGCTGGCTCTCCAA TSESATPES CTTCTACTGAAGAAGGTAGCCC GPGTSESATGGCAGGCTCTCCGACCTCTACT PESGPGSEP GAGGAAGGTACTTCTGAAAGCG ATSGSETPGCAACCCCGGAGTCCGGCCCAGG SEPATSGSE TACCTCTACCGAACCGTCTGAG TPGSPAGSPGGCAGCGCACCAGGTACCTCTG TSTEEGTST AAAGCGCAACTCCTGAGTCTGG EPSEGSAPGCCCAGGTAGCGAACCTGCTACC TSTEPSEGS TCCGGCTCTGAGACTCCAGGTA APGSEPATSCCTCTGAAAGCGCAACCCCGGA GSETPGTSE ATCTGGTCCAGGTAGCGAACCT SATPESGPGGCAACCTCTGGCTCTGAAACCC TSTEPSEGS CAGGTACCTCTGAAAGCGCTAC APTCCTGAATCTGGCCCAGGTACT TCTACTGAACCGTCCGAGGGCA GCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA GGTACCTCTGAAAGCGCAACCC CTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA ACCCCAGGTACTTCTGAAAGCG CTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT TCCACCGAGGAAGGTAGCCCGG CTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCT TCCGAGGGCAGCGCACCAGGTA CTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA AGCGCTACTCCTGAATCCGGTC CAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC GAACCGGCTACTTCTGGTTCTG AAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA GGTAGCCCAGCAGGCTCTCCGA CTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGC GCACCAGGTACCTCTACTGAAC CTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC TCTGAAACCCCAGGTACCTCTG AAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCG TCCGAGGGCAGCGCACCA FVII- ANAFLEELR 726GCCAACGCGTTCCTGGAGGAGC 727 FIIa- PGSLERECK TACGGCCGGGCTCCCTGGAGAG AE288EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAER CTCCTTCGAGGAGGCCCGGGAGTKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTACCQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGGLPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCCCVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAGKRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAGCTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGTNASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAAECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAGTLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAAKNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCADGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCGVPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATCVLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATGRTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTADRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGCTQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTGEYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTTDSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCAIVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACCRVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAAPGVLLRAPF ATATCACGGAGTACATGTTCTG PGLTPRSLL TGCCGGCTACTCGGATGGCAGCVGGTSESAT AAGGACTCCTGCAAGGGGGAC PESGPGSEP AGTGGAGGCCCACATGCCACCCATSGSETPG ACTACCGGGGCACGTGGTACCT TSESATPES GACGGGCATCGTCAGCTGGGGCGPGSEPATS CAGGGCTGCGCAACCGTGGGCC GSETPGTSE ACTTTGGGGTGTACACCAGGGTSATPESGPG CTCCCAGTACATCGAGTGGCTG TSTEPSEGS CAAAAGCTCATGCGCTCAGAGCAPGSPAGSP CACGCCCAGGAGTCCTCCTGCG TSTEEGTSE AGCCCCATTTCCCGGT0GGTGGTSATPESGPG ACCTCTGAAAGCGCAACTCCTG SEPATSGSE AGTCTGGCCCAGGTAGCGAACCTPGTSESAT TGCTACCTCCGGCTCTGAGACT PESGPGSPA CCAGGTACCTCTGAAAGCGCAAGSPTSTEEG CCCCGGAATCTGGTCCAGGTAG SPAGSPTST CGAACCTGCAACCTCTGGCTCTEEGTSTEPS GAAACCCCAGGTACCTCTGAAA EGSAPGTSE GCGCTACTCCTGAATCTGGCCCSATPESGPG AGGTACTTCTACTGAACCGTCC TSESATPES GAGGGCAGCGCACCAGGTAGCGPGTSESAT CCTGCTGGCTCTCCAACCTCCA PESGPGSEP CCGAAGAAGGTACCTCTGAAAGATSGSETPG CGCAACCCCTGAATCCGGCCCA SEPATSGSE GGTAGCGAACCGGCAACCTCCGTPGSPAGSP GTTCTGAAACCCCAGGTACTTC TSTEEGTST TGAAAGCGCTACTCCTGAGTCCEPSEGSAPG GGCCCAGGTAGCCCGGCTGGCT TSTEPSEGS CTCCGACTTCCACCGAGGAAGGAPGSEPATS TAGCCCGGCTGGCTCTCCAACT GSETPGTSE TCTACTGAAGAAGGTACTTCTASATPESGPG CCGAACCTTCCGAGGGCAGCGC TSTEPSEGS ACCAGGTACTTCTGAAAGCGCT APACCCCTGAGTCCGGCCCAGGTA CTTCTGAAAGCGCTACTCCTGA ATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCC CAGGTAGCGAACCGGCTACTTC TGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTG AAACTCCAGGTAGCCCAGCAGG CTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG AAGGCAGCGCACCAGGTACCTC TACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGG TACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGC ACCA FVII- ANAFLEELR 728GCCAACGCGTTCCTGGAGGAGC 729 FIIa- PGSLERECK TACGGCCGGGCTCCCTGGAGAG AE864EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG REIFKDAER CTCCTTCGAGGAGGCCCGGGAGTKLFWISYS ATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTACCQNGGSCKD AGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGGLPAFEGRNC CTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCCCVNENGGCE TGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAGKRSCRCHEG CTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAGCTPTVEYPC TGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGTNASKPQGRI ACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAAECPWQVLLL TATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAGTLINTIWVV CAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAAKNWRNLIAV GGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCADGDEQSRRV GTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCGVPGTTNHDI CGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATCVLTDHVVPL GCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATGRTLAFVRFS AGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTADRGATALEL CGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGCTQDCLQQSR ACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTGEYMFCAGYS CCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTTDSGGPHATH CTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCAIVSWGQGCA CGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACCRVSQYIEWL CAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAAPGVLLRAPF ATATCACGGAGTACATGTTCTG PGLTPRSLL TGCCGGCTACTCGGATGGCAGCVGGSPAGSP AAGGACTCCTGCAAGGGGGAC TSTEEGTSE AGTGGAGGCCCACATGCCACCCSATPESGPG ACTACCGGGGCACGTGGTACCT TSTEPSEGS GACGGGCATCGTCAGCTGGGGCAPGSPAGSP CAGGGCTGCGCAACCGTGGGCC TSTEEGTST ACTTTGGGGTGTACACCAGGGTEPSEGSAPG CTCCCAGTACATCGAGTGGCTG TSTEPSEGS CAAAAGCTCATGCGCTCAGAGCAPGTSESAT CACGCCCAGGAGTCCTCCTGCG PESGPGSEP AGCCCCATTTCCCGGT0GGTGGTATSGSETPG AGCCCGGCTGGCTCTCCTACCT SEPATSGSE CTACTGAGGAAGGTACTTCTGATPGSPAGSP AAGCGCTACTCCTGAGTCTGGT TSTEEGTSE CCAGGTACCTCTACTGAACCGTSATPESGPG CCGAAGGTAGCGCTCCAGGTAG TSTEPSEGS CCCAGCAGGCTCTCCGACTTCCAPGTSTEPS ACTGAGGAAGGTACTTCTACTG EGSAPGSPA AACCTTCCGAAGGCAGCGCACCGSPTSTEEG AGGTACCTCTACTGAACCTTCT TSTEPSEGS GAGGGCAGCGCTCCAGGTACTTAPGTSTEPS CTGAAAGCGCTACCCCGGAATC EGSAPGTSE TGGCCCAGGTAGCGAACCGGCTSATPESGPG ACTTCTGGTTCTGAAACCCCAG TSTEPSEGS GTAGCGAACCGGCTACCTCCGGAPGTSESAT TTCTGAAACTCCAGGTAGCCCG PESGPGSEP GCAGGCTCTCCGACCTCTACTGATSGSETPG AGGAAGGTACTTCTGAAAGCGC TSTEPSEGS AACCCCGGAGTCCGGCCCAGGTAPGTSTEPS ACCTCTACCGAACCGTCTGAGG EGSAPGTSE GCAGCGCACCAGGTACTTCTACSATPESGPG CGAACCGTCCGAGGGTAGCGCA TSESATPES CCAGGTAGCCCAGCAGGTTCTCGPGSPAGSP CTACCTCCACCGAGGAAGGTAC TSTEEGTSE TTCTACCGAACCGTCCGAGGGTSATPESGPG AGCGCACCAGGTACCTCTACTG SEPATSGSE AACCTTCTGAGGGCAGCGCTCCTPGTSESAT AGGTACTTCTGAAAGCGCTACC PESGPGTST CCGGAGTCCGGTCCAGGTACTTEPSEGSAPG CTACTGAACCGTCCGAAGGTAG TSTEPSEGS CGCACCAGGTACTTCTGAAAGCAPGTSTEPS GCAACCCCTGAATCCGGTCCAG EGSAPGTST GTAGCGAACCGGCTACTTCTGGEPSEGSAPG CTCTGAGACTCCAGGTACTTCT TSTEPSEGS ACCGAACCGTCCGAAGGTAGCGAPGTSTEPS CACCAGGTACTTCTACTGAACC EGSAPGSPA GTCTGAAGGTAGCGCACCAGGTGSPTSTEEG ACTTCTGAAAGCGCAACCCCGG TSTEPSEGS AATCCGGCCCAGGTACCTCTGAAPGTSESAT AAGCGCAACCCCGGAGTCCGGC PESGPGSEP CCAGGTAGCCCTGCTGGCTCTCATSGSETPG CAACCTCCACCGAAGAAGGTAC TSESATPES CTCTGAAAGCGCAACCCCTGAAGPGSEPATS TCCGGCCCAGGTAGCGAACCGG GSETPGTSE CAACCTCCGGTTCTGAAACCCCSATPESGPG AGGTACCTCTGAAAGCGCTACT TSTEPSEGS CCGGAGTCTGGCCCAGGTACCTAPGTSESAT CTACTGAACCGTCTGAGGGTAG PESGPGSPA CGCTCCAGGTACTTCTACTGAAGSPTSTEEG CCGTCCGAAGGTAGCGCACCAG SPAGSPTST GTACTTCTACCGAACCGTCCGAEEGSPAGSP AGGCAGCGCTCCAGGTACCTCT TSTEEGTSE ACTGAACCTTCCGAGGGCAGCGSATPESGPG CTCCAGGTACCTCTACCGAACC TSTEPSEGS TTCTGAAGGTAGCGCACCAGGTAPGTSESAT ACTTCTACCGAACCGTCCGAGG PESGPGSEP GTAGCGCACCAGGTAGCCCAGCATSGSETPG AGGTTCTCCTACCTCCACCGAG TSESATPES GAAGGTACTTCTACCGAACCGTGPGSEPATS CCGAGGGTAGCGCACCAGGTAC GSETPGTSE CTCTGAAAGCGCAACTCCTGAGSATPESGPG TCTGGCCCAGGTAGCGAACCTG TSTEPSEGS CTACCTCCGGCTCTGAGACTCCAPGSPAGSP AGGTACCTCTGAAAGCGCAACC TSTEEGTSE CCGGAATCTGGTCCAGGTAGCGSATPESGPG AACCTGCAACCTCTGGCTCTGA SEPATSGSE AACCCCAGGTACCTCTGAAAGCTPGTSESAT GCTACTCCTGAATCTGGCCCAG PESGPGSPA GTACTTCTACTGAACCGTCCGAGSPTSTEEG GGGCAGCGCACCAGGTACTTCT SPAGSPTST GAAAGCGCTACTCCTGAGTCCGEEGTSTEPS GCCCAGGTAGCCCGGCTGGCTC EGSAPGTSE TCCGACTTCCACCGAGGAAGGTSATPESGPG AGCCCGGCTGGCTCTCCAACTT TSESATPES CTACTGAAGAAGGTAGCCCGGCGPGTSESAT AGGCTCTCCGACCTCTACTGAG PESGPGSEP GAAGGTACTTCTGAAAGCGCAAATSGSETPG CCCCGGAGTCCGGCCCAGGTAC SEPATSGSE CTCTACCGAACCGTCTGAGGGCTPGSPAGSP AGCGCACCAGGTACCTCTGAAA TSTEEGTST GCGCAACTCCTGAGTCTGGCCCEPSEGSAPG AGGTAGCGAACCTGCTACCTCC TSTEPSEGS GGCTCTGAGACTCCAGGTACCTAPGSEPATS CTGAAAGCGCAACCCCGGAATC GSETPGTSE TGGTCCAGGTAGCGAACCTGCASATPESGPG ACCTCTGGCTCTGAAACCCCAG TSTEPSEGS GTACCTCTGAAAGCGCTACTCC APTGAATCTGGCCCAGGTACTTCT ACTGAACCGTCCGAGGGCAGCG CACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGT ACCTCTGAAAGCGCAACCCCTG AATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTA CTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTG GCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTT CTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAG GTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAA CCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTT CCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTT CTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAA GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA FIX- YNSGKLEEF 730TATAATTCAGGTAAATTGGAAG 731 FIIa- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA AE288MEEKCSFEE GAGAGAATGTATGGAAGAAAA AREVFENTE GTGTAGTTTTGAAGAAGCACGARTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCAPCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATGCPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTCKNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGTCSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGCAVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTGRAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGTTILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGAVVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGANGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATGVTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCAEHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTGIIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCCALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAACIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATGVSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATAYLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTATIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGACQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTGLTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTTIYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCGTGLTPRSLL ATCTACAAAGTTCACCATCTAT VGGTSESAT AACAACATGTTCTGTGCTGGCTPESGPGSEP TCCATGAAGGAGGTAGAGATTC ATSGSETPG ATGTCAAGGAGATAGTGGGGGTSESATPES ACCCCATGTTACTGAAGTGGAA GPGSEPATS GGGACCAGTTTCTTAACTGGAAGSETPGTSE TTATTAGCTGGGGTGAAGAGTG SATPESGPG TGCAATGAAAGGCAAATATGGATSTEPSEGS ATATATACCAAGGTATCCCGGT APGSPAGSP ATGTCAACTGGATTAAGGAAAATSTEEGTSE AACAAAGCTCACTGGGGT0GGT SATPESGPG GGTACCTCTGAAAGCGCAACTCSEPATSGSE CTGAGTCTGGCCCAGGTAGCGA TPGTSESAT ACCTGCTACCTCCGGCTCTGAGPESGPGSPA ACTCCAGGTACCTCTGAAAGCG GSPTSTEEG CAACCCCGGAATCTGGTCCAGGSPAGSPTST TAGCGAACCTGCAACCTCTGGC EEGTSTEPS TCTGAAACCCCAGGTACCTCTGEGSAPGTSE AAAGCGCTACTCCTGAATCTGG SATPESGPG CCCAGGTACTTCTACTGAACCGTSESATPES TCCGAGGGCAGCGCACCAGGTA GPGTSESAT GCCCTGCTGGCTCTCCAACCTCPESGPGSEP CACCGAAGAAGGTACCTCTGAA ATSGSETPG AGCGCAACCCCTGAATCCGGCCSEPATSGSE CAGGTAGCGAACCGGCAACCTC TPGSPAGSP CGGTTCTGAAACCCCAGGTACTTSTEEGTST TCTGAAAGCGCTACTCCTGAGT EPSEGSAPG CCGGCCCAGGTAGCCCGGCTGGTSTEPSEGS CTCTCCGACTTCCACCGAGGAA APGSEPATS GGTAGCCCGGCTGGCTCTCCAAGSETPGTSE CTTCTACTGAAGAAGGTACTTC SATPESGPG TACCGAACCTTCCGAGGGCAGCTSTEPSEGS GCACCAGGTACTTCTGAAAGCG AP CTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT GAATCCGGTCCAGGTACTTCTG AAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT TCTGGTTCTGAAACCCCAGGTA GCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA GGCTCTCCGACTTCCACTGAGG AAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACC TCTACTGAACCTTCTGAGGGCA GCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA GGTACCTCTGAAAGCGCTACTC CTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FIX- YNSGKLEEF 732 TATAATTCAGGTAAATTGGAAG733 FIIa- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA AE864 MEEKCSFEEGAGAGAATGTATGGAAGAAAA AREVFENTE GTGTAGTTTTGAAGAAGCACGA RTTEFWKQYGAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCA PCLNGGSCKGTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATG CPFGFEGKNGCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTC KNGRCEQFCCCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGT CSCTEGYRLAACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGC AVPFPCGRVTGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTG RAETVFPDVCAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGT TILDNITQSGGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGA VVGGEDAKPGACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGA NGKVDAFCGAACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATG VTAAHCVETACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCA EHNIEETEHATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTG IIPHHNYNATGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCC ALLELDEPLACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAA CIADKEYTNCATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATG VSGWGRVFHTGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATA YLRVPLVDRAGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTA TIYNNMFCAGTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGA CQGDSGGPHATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTG LTGIISWGEGCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTT IYTKVSRYVCAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCG TGLTPRSLLATCTACAAAGTTCACCATCTAT VGGSPAGSP AACAACATGTTCTGTGCTGGCT TSTEEGTSETCCATGAAGGAGGTAGAGATTC SATPESGPG ATGTCAAGGAGATAGTGGGGG TSTEPSEGSACCCCATGTTACTGAAGTGGAA APGSPAGSP GGGACCAGTTTCTTAACTGGAA TSTEEGTSTTTATTAGCTGGGGTGAAGAGTG EPSEGSAPG TGCAATGAAAGGCAAATATGGA TSTEPSEGSATATATACCAAGGTATCCCGGT APGTSESAT ATGTCAACTGGATTAAGGAAAA PESGPGSEPAACAAAGCTCACTGGGGT0GGT ATSGSETPG GGTAGCCCGGCTGGCTCTCCTA SEPATSGSECCTCTACTGAGGAAGGTACTTC TPGSPAGSP TGAAAGCGCTACTCCTGAGTCT TSTEEGTSEGGTCCAGGTACCTCTACTGAAC SATPESGPG CGTCCGAAGGTAGCGCTCCAGG TSTEPSEGSTAGCCCAGCAGGCTCTCCGACT APGTSTEPS TCCACTGAGGAAGGTACTTCTA EGSAPGSPACTGAACCTTCCGAAGGCAGCGC GSPTSTEEG ACCAGGTACCTCTACTGAACCT TSTEPSEGSTCTGAGGGCAGCGCTCCAGGTA APGTSTEPS CTTCTGAAAGCGCTACCCCGGA EGSAPGTSEATCTGGCCCAGGTAGCGAACCG SATPESGPG GCTACTTCTGGTTCTGAAACCC TSTEPSEGSCAGGTAGCGAACCGGCTACCTC APGTSESAT CGGTTCTGAAACTCCAGGTAGC PESGPGSEPCCGGCAGGCTCTCCGACCTCTA ATSGSETPG CTGAGGAAGGTACTTCTGAAAG TSTEPSEGSCGCAACCCCGGAGTCCGGCCCA APGTSTEPS GGTACCTCTACCGAACCGTCTG EGSAPGTSEAGGGCAGCGCACCAGGTACTTC SATPESGPG TACCGAACCGTCCGAGGGTAGC TSESATPESGCACCAGGTAGCCCAGCAGGTT GPGSPAGSP CTCCTACCTCCACCGAGGAAGG TSTEEGTSETACTTCTACCGAACCGTCCGAG SATPESGPG GGTAGCGCACCAGGTACCTCTA SEPATSGSECTGAACCTTCTGAGGGCAGCGC TPGTSESAT TCCAGGTACTTCTGAAAGCGCT PESGPGTSTACCCCGGAGTCCGGTCCAGGTA EPSEGSAPG CTTCTACTGAACCGTCCGAAGG TSTEPSEGSTAGCGCACCAGGTACTTCTGAA APGTSTEPS AGCGCAACCCCTGAATCCGGTC EGSAPGTSTCAGGTAGCGAACCGGCTACTTC EPSEGSAPG TGGCTCTGAGACTCCAGGTACT TSTEPSEGSTCTACCGAACCGTCCGAAGGTA APGTSTEPS GCGCACCAGGTACTTCTACTGA EGSAPGSPAACCGTCTGAAGGTAGCGCACCA GSPTSTEEG GGTACTTCTGAAAGCGCAACCC TSTEPSEGSCGGAATCCGGCCCAGGTACCTC APGTSESAT TGAAAGCGCAACCCCGGAGTCC PESGPGSEPGGCCCAGGTAGCCCTGCTGGCT ATSGSETPG CTCCAACCTCCACCGAAGAAGG TSESATPESTACCTCTGAAAGCGCAACCCCT GPGSEPATS GAATCCGGCCCAGGTAGCGAAC GSETPGTSECGGCAACCTCCGGTTCTGAAAC SATPESGPG CCCAGGTACCTCTGAAAGCGCT TSTEPSEGSACTCCGGAGTCTGGCCCAGGTA APGTSESAT CCTCTACTGAACCGTCTGAGGG PESGPGSPATAGCGCTCCAGGTACTTCTACT GSPTSTEEG GAACCGTCCGAAGGTAGCGCAC SPAGSPTSTCAGGTACTTCTACCGAACCGTC EEGSPAGSP CGAAGGCAGCGCTCCAGGTACC TSTEEGTSETCTACTGAACCTTCCGAGGGCA SATPESGPG GCGCTCCAGGTACCTCTACCGA TSTEPSEGSACCTTCTGAAGGTAGCGCACCA APGTSESAT GGTACTTCTACCGAACCGTCCG PESGPGSEPAGGGTAGCGCACCAGGTAGCCC ATSGSETPG AGCAGGTTCTCCTACCTCCACC TSESATPESGAGGAAGGTACTTCTACCGAAC GPGSEPATS CGTCCGAGGGTAGCGCACCAGG GSETPGTSETACCTCTGAAAGCGCAACTCCT SATPESGPG GAGTCTGGCCCAGGTAGCGAAC TSTEPSEGSCTGCTACCTCCGGCTCTGAGAC APGSPAGSP TCCAGGTACCTCTGAAAGCGCA TSTEEGTSEACCCCGGAATCTGGTCCAGGTA SATPESGPG GCGAACCTGCAACCTCTGGCTC SEPATSGSETGAAACCCCAGGTACCTCTGAA TPGTSESAT AGCGCTACTCCTGAATCTGGCC PESGPGSPACAGGTACTTCTACTGAACCGTC GSPTSTEEG CGAGGGCAGCGCACCAGGTACT SPAGSPTSTTCTGAAAGCGCTACTCCTGAGT EEGTSTEPS CCGGCCCAGGTAGCCCGGCTGG EGSAPGTSECTCTCCGACTTCCACCGAGGAA SATPESGPG GGTAGCCCGGCTGGCTCTCCAA TSESATPESCTTCTACTGAAGAAGGTAGCCC GPGTSESAT GGCAGGCTCTCCGACCTCTACT PESGPGSEPGAGGAAGGTACTTCTGAAAGCG ATSGSETPG CAACCCCGGAGTCCGGCCCAGG SEPATSGSETACCTCTACCGAACCGTCTGAG TPGSPAGSP GGCAGCGCACCAGGTACCTCTG TSTEEGTSTAAAGCGCAACTCCTGAGTCTGG EPSEGSAPG CCCAGGTAGCGAACCTGCTACC TSTEPSEGSTCCGGCTCTGAGACTCCAGGTA APGSEPATS CCTCTGAAAGCGCAACCCCGGA GSETPGTSEATCTGGTCCAGGTAGCGAACCT SATPESGPG GCAACCTCTGGCTCTGAAACCC TSTEPSEGSCAGGTACCTCTGAAAGCGCTAC AP TCCTGAATCTGGCCCAGGTACT TCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGG CTCTCCAACCTCCACCGAAGAA GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGA ACCGGCAACCTCCGGTTCTGAA ACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGG TAGCCCGGCTGGCTCTCCGACT TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGA AGAAGGTACTTCTACCGAACCT TCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGA GTCCGGCCCAGGTACTTCTGAA AGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC CCCGGAATCTGGCCCAGGTAGC GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGC TACCTCCGGTTCTGAAACTCCA GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTC TACTGAACCTTCCGAAGGCAGC GCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG TAGCGAACCTGCAACCTCTGGC TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGG CCCAGGTACTTCTACTGAACCG TCCGAGGGCAGCGCACCA FVII-ANAFLEELR 734 GCCAACGCGTTCCTGGAGGAGC 735 MMP- PGSLERECKTACGGCCGGGCTCCCTGGAGAG 17- EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG AE288REIFKDAER CTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGADGDQCASSP CGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCTQLQSYICFC CAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAGETHKDDQLI TCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGTQYCSDHTGT GAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACGYSLLADGVS GCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGCGKIPILEKR TCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGTVGGKVCPKG GTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTAVNGAQLCGG TTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTGSAAHCFDKI GGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCCLGEHDLSEH TGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATCAQVIIPSTY AACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAATALLRLHQPV CAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACCCLPERTFSE TCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGCLVSGWGQLL AGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCACMVLNVPRLM GACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGAKVGDSPNIT CCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGADGSKDSCKG GGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGCYRGTWYLTG CAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCTTVGHFGVYT CAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCACQKLMRSEPR GGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTGPGAPLGLRL TGCCGGCTACTCGGATGGCAGC RGGTSESAT AAGGACTCCTGCAAGGGGGACPESGPGSEP AGTGGAGGCCCACATGCCACCC ATSGSETPG ACTACCGGGGCACGTGGTACCTTSESATPES GACGGGCATCGTCAGCTGGGGC GPGSEPATS CAGGGCTGCGCAACCGTGGGCCGSETPGTSE ACTTTGGGGTGTACACCAGGGT SATPESGPG CTCCCAGTACATCGAGTGGCTGTSTEPSEGS CAAAAGCTCATGCGCTCAGAGC APGSPAGSP CACGCCCAGGAGTCCTCCTGCGTSTEEGTSE AGCCCCATTTCCCGGT0GGTGGT SATPESGPG ACCTCTGAAAGCGCAACTCCTGSEPATSGSE AGTCTGGCCCAGGTAGCGAACC TPGTSESAT TGCTACCTCCGGCTCTGAGACTPESGPGSPA CCAGGTACCTCTGAAAGCGCAA GSPTSTEEG CCCCGGAATCTGGTCCAGGTAGSPAGSPTST CGAACCTGCAACCTCTGGCTCT EEGTSTEPS GAAACCCCAGGTACCTCTGAAAEGSAPGTSE GCGCTACTCCTGAATCTGGCCC SATPESGPG AGGTACTTCTACTGAACCGTCCTSESATPES GAGGGCAGCGCACCAGGTAGC GPGTSESAT CCTGCTGGCTCTCCAACCTCCAPESGPGSEP CCGAAGAAGGTACCTCTGAAAG ATSGSETPG CGCAACCCCTGAATCCGGCCCASEPATSGSE GGTAGCGAACCGGCAACCTCCG TPGSPAGSP GTTCTGAAACCCCAGGTACTTCTSTEEGTST TGAAAGCGCTACTCCTGAGTCC EPSEGSAPG GGCCCAGGTAGCCCGGCTGGCTTSTEPSEGS CTCCGACTTCCACCGAGGAAGG APGSEPATS TAGCCCGGCTGGCTCTCCAACTGSETPGTSE TCTACTGAAGAAGGTACTTCTA SATPESGPG CCGAACCTTCCGAGGGCAGCGCTSTEPSEGS ACCAGGTACTTCTGAAAGCGCT AP ACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGA ATCCGGTCCAGGTACTTCTGAA AGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTC TGGTTCTGAAACCCCAGGTAGC GAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGG CTCTCCGACTTCCACTGAGGAA GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTC TACTGAACCTTCTGAGGGCAGC GCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG TACCTCTGAAAGCGCTACTCCT GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGC ACCA FVII- ANAFLEELR 736 GCCAACGCGTTCCTGGAGGAGC737 MMP- PGSLERECK TACGGCCGGGCTCCCTGGAGAG 17- EEQCSFEEAGGAGTGCAAGGAGGAGCAGTG AE864 REIFKDAER CTCCTTCGAGGAGGCCCGGGAG TKLFWISYSATCTTCAAGGACGCGGAGAGGA DGDQCASSP CGAAGCTGTTCTGGATTTCTTAC CQNGGSCKDAGTGATGGGGACCAGTGTGCCT QLQSYICFC CAAGTCCATGCCAGAATGGGGG LPAFEGRNCCTCCTGCAAGGACCAGCTCCAG ETHKDDQLI TCCTATATCTGCTTCTGCCTCCC CVNENGGCETGCCTTCGAGGGCCGGAACTGT QYCSDHTGT GAGACGCACAAGGATGACCAG KRSCRCHEGCTGATCTGTGTGAACGAGAACG YSLLADGVS GCGGCTGTGAGCAGTACTGCAG CTPTVEYPCTGACCACACGGGCACCAAGCGC GKIPILEKR TCCTGTCGGTGCCACGAGGGGT NASKPQGRIACTCTCTGCTGGCAGACGGGGT VGGKVCPKG GTCCTGCACACCCACAGTTGAA ECPWQVLLLTATCCATGTGGAAAAATACCTA VNGAQLCGG TTCTAGAAAAAAGAAATGCCAG TLINTIWVVCAAACCCCAAGGCCGAATTGTG SAAHCFDKI GGGGGCAAGGTGTGCCCCAAA KNWRNLIAVGGGGAGTGTCCATGGCAGGTCC LGEHDLSEH TGTTGTTGGTGAATGGAGCTCA DGDEQSRRVGTTGTGTGGGGGGACCCTGATC AQVIIPSTY AACACCATCTGGGTGGTCTCCG VPGTTNHDICGGCCCACTGTTTCGACAAAAT ALLRLHQPV CAAGAACTGGAGGAACCTGATC VLTDHVVPLGCGGTGCTGGGCGAGCACGACC CLPERTFSE TCAGCGAGCACGACGGGGATG RTLAFVRFSAGCAGAGCCGGCGGGTGGCGC LVSGWGQLL AGGTCATCATCCCCAGCACGTA DRGATALELCGTCCCGGGCACCACCAACCAC MVLNVPRLM GACATCGCGCTGCTCCGCCTGC TQDCLQQSRACCAGCCCGTGGTCCTCACTGA KVGDSPNIT CCATGTGGTGCCCCTCTGCCTG EYMFCAGYSCCCGAACGGACGTTCTCTGAGA DGSKDSCKG GGACGCTGGCCTTCGTGCGCTT DSGGPHATHCTCATTGGTCAGCGGCTGGGGC YRGTWYLTG CAGCTGCTGGACCGTGGCGCCA IVSWGQGCACGGCCCTGGAGCTCATGGTCCT TVGHFGVYT CAACGTGCCCCGGCTGATGACC RVSQYIEWLCAGGACTGCCTGCAGCAGTCAC QKLMRSEPR GGAAGGTGGGAGACTCCCCAA PGVLLRAPFATATCACGGAGTACATGTTCTG PGAPLGLRL TGCCGGCTACTCGGATGGCAGC RGGSPAGSPAAGGACTCCTGCAAGGGGGAC TSTEEGTSE AGTGGAGGCCCACATGCCACCC SATPESGPGACTACCGGGGCACGTGGTACCT TSTEPSEGS GACGGGCATCGTCAGCTGGGGC APGSPAGSPCAGGGCTGCGCAACCGTGGGCC TSTEEGTST ACTTTGGGGTGTACACCAGGGT EPSEGSAPGCTCCCAGTACATCGAGTGGCTG TSTEPSEGS CAAAAGCTCATGCGCTCAGAGC APGTSESATCACGCCCAGGAGTCCTCCTGCG PESGPGSEP AGCCCCATTTCCCGGT0GGTGGT ATSGSETPGAGCCCGGCTGGCTCTCCTACCT SEPATSGSE CTACTGAGGAAGGTACTTCTGA TPGSPAGSPAAGCGCTACTCCTGAGTCTGGT TSTEEGTSE CCAGGTACCTCTACTGAACCGT SATPESGPGCCGAAGGTAGCGCTCCAGGTAG TSTEPSEGS CCCAGCAGGCTCTCCGACTTCC APGTSTEPSACTGAGGAAGGTACTTCTACTG EGSAPGSPA AACCTTCCGAAGGCAGCGCACC GSPTSTEEGAGGTACCTCTACTGAACCTTCT TSTEPSEGS GAGGGCAGCGCTCCAGGTACTT APGTSTEPSCTGAAAGCGCTACCCCGGAATC EGSAPGTSE TGGCCCAGGTAGCGAACCGGCT SATPESGPGACTTCTGGTTCTGAAACCCCAG TSTEPSEGS GTAGCGAACCGGCTACCTCCGG APGTSESATTTCTGAAACTCCAGGTAGCCCG PESGPGSEP GCAGGCTCTCCGACCTCTACTG ATSGSETPGAGGAAGGTACTTCTGAAAGCGC TSTEPSEGS AACCCCGGAGTCCGGCCCAGGT APGTSTEPSACCTCTACCGAACCGTCTGAGG EGSAPGTSE GCAGCGCACCAGGTACTTCTAC SATPESGPGCGAACCGTCCGAGGGTAGCGCA TSESATPES CCAGGTAGCCCAGCAGGTTCTC GPGSPAGSPCTACCTCCACCGAGGAAGGTAC TSTEEGTSE TTCTACCGAACCGTCCGAGGGT SATPESGPGAGCGCACCAGGTACCTCTACTG SEPATSGSE AACCTTCTGAGGGCAGCGCTCC TPGTSESATAGGTACTTCTGAAAGCGCTACC PESGPGTST CCGGAGTCCGGTCCAGGTACTT EPSEGSAPGCTACTGAACCGTCCGAAGGTAG TSTEPSEGS CGCACCAGGTACTTCTGAAAGC APGTSTEPSGCAACCCCTGAATCCGGTCCAG EGSAPGTST GTAGCGAACCGGCTACTTCTGG EPSEGSAPGCTCTGAGACTCCAGGTACTTCT TSTEPSEGS ACCGAACCGTCCGAAGGTAGCG APGTSTEPSCACCAGGTACTTCTACTGAACC EGSAPGSPA GTCTGAAGGTAGCGCACCAGGT GSPTSTEEGACTTCTGAAAGCGCAACCCCGG TSTEPSEGS AATCCGGCCCAGGTACCTCTGA APGTSESATAAGCGCAACCCCGGAGTCCGGC PESGPGSEP CCAGGTAGCCCTGCTGGCTCTC ATSGSETPGCAACCTCCACCGAAGAAGGTAC TSESATPES CTCTGAAAGCGCAACCCCTGAA GPGSEPATSTCCGGCCCAGGTAGCGAACCGG GSETPGTSE CAACCTCCGGTTCTGAAACCCC SATPESGPGAGGTACCTCTGAAAGCGCTACT TSTEPSEGS CCGGAGTCTGGCCCAGGTACCT APGTSESATCTACTGAACCGTCTGAGGGTAG PESGPGSPA CGCTCCAGGTACTTCTACTGAA GSPTSTEEGCCGTCCGAAGGTAGCGCACCAG SPAGSPTST GTACTTCTACCGAACCGTCCGA EEGSPAGSPAGGCAGCGCTCCAGGTACCTCT TSTEEGTSE ACTGAACCTTCCGAGGGCAGCG SATPESGPGCTCCAGGTACCTCTACCGAACC TSTEPSEGS TTCTGAAGGTAGCGCACCAGGT APGTSESATACTTCTACCGAACCGTCCGAGG PESGPGSEP GTAGCGCACCAGGTAGCCCAGC ATSGSETPGAGGTTCTCCTACCTCCACCGAG TSESATPES GAAGGTACTTCTACCGAACCGT GPGSEPATSCCGAGGGTAGCGCACCAGGTAC GSETPGTSE CTCTGAAAGCGCAACTCCTGAG SATPESGPGTCTGGCCCAGGTAGCGAACCTG TSTEPSEGS CTACCTCCGGCTCTGAGACTCC APGSPAGSPAGGTACCTCTGAAAGCGCAACC TSTEEGTSE CCGGAATCTGGTCCAGGTAGCG SATPESGPGAACCTGCAACCTCTGGCTCTGA SEPATSGSE AACCCCAGGTACCTCTGAAAGC TPGTSESATGCTACTCCTGAATCTGGCCCAG PESGPGSPA GTACTTCTACTGAACCGTCCGA GSPTSTEEGGGGCAGCGCACCAGGTACTTCT SPAGSPTST GAAAGCGCTACTCCTGAGTCCG EEGTSTEPSGCCCAGGTAGCCCGGCTGGCTC EGSAPGTSE TCCGACTTCCACCGAGGAAGGT SATPESGPGAGCCCGGCTGGCTCTCCAACTT TSESATPES CTACTGAAGAAGGTAGCCCGGC GPGTSESATAGGCTCTCCGACCTCTACTGAG PESGPGSEP GAAGGTACTTCTGAAAGCGCAA ATSGSETPGCCCCGGAGTCCGGCCCAGGTAC SEPATSGSE CTCTACCGAACCGTCTGAGGGC TPGSPAGSPAGCGCACCAGGTACCTCTGAAA TSTEEGTST GCGCAACTCCTGAGTCTGGCCC EPSEGSAPGAGGTAGCGAACCTGCTACCTCC TSTEPSEGS GGCTCTGAGACTCCAGGTACCT APGSEPATSCTGAAAGCGCAACCCCGGAATC GSETPGTSE TGGTCCAGGTAGCGAACCTGCA SATPESGPGACCTCTGGCTCTGAAACCCCAG TSTEPSEGS GTACCTCTGAAAGCGCTACTCC APTGAATCTGGCCCAGGTACTTCT ACTGAACCGTCCGAGGGCAGCG CACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGT ACCTCTGAAAGCGCAACCCCTG AATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTA CTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTG GCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTT CTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAG GTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAA CCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTT CCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTT CTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAA GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA FIX- YNSGKLEEF 738TATAATTCAGGTAAATTGGAAG 739 MMP- VQGNLEREC AGTTTGTTCAAGGGAACCTTGA 17-MEEKCSFEE GAGAGAATGTATGGAAGAAAA FXIa AREVFENTE GTGTAGTTTTGAAGAAGCACGARTTEFWKQY GAAGTTTTTGAAAACACTGAAA VDGDQCESN GAACAACTGAATTTTGGAAGCAPCLNGGSCK GTATGTTGATGGAGATCAGTGT DDINSYECW GAGTCCAATCCATGTTTAAATGCPFGFEGKN GCGGCAGTTGCAAGGATGACAT CELDVTCNI TAATTCCTATGAATGTTGGTGTCKNGRCEQFC CCTTTGGATTTGAAGGAAAGAA KNSADNKVV CTGTGAATTAGATGTAACATGTCSCTEGYRL AACATTAAGAATGGCAGATGCG AENQKSCEP AGCAGTTTTGTAAAAATAGTGCAVPFPCGRV TGATAACAAGGTGGTTTGCTCC SVSQTSKLT TGTACTGAGGGATATCGACTTGRAETVFPDV CAGAAAACCAGAAGTCCTGTGA DYVNSTEAE ACCAGCAGTGCCATTTCCATGTTILDNITQS GGAAGAGTTTCTGTTTCACAAA TQSFNDFTR CTTCTAAGCTCACCCGTGCTGAVVGGEDAKP GACTGTTTTTCCTGATGTGGACT GQFPWQVVL ATGTAAATTCTACTGAAGCTGANGKVDAFCG AACCATTTTGGATAACATCACT GSIVNEKWI CAAAGCACCCAATCATTTAATGVTAAHCVET ACTTCACTCGGGTTGTTGGTGG GVKITVVAG AGAAGATGCCAAACCAGGTCAEHNIEETEH ATTCCCTTGGCAGGTTGTTTTGA TEQKRNVIR ATGGTAAAGTTGATGCATTCTGIIPHHNYNA TGGAGGCTCTATCGTTAATGAA AINKYNHDI AAATGGATTGTAACTGCTGCCCALLELDEPL ACTGTGTTGAAACTGGTGTTAA VLNSYVTPI AATTACAGTTGTCGCAGGTGAACIADKEYTN CATAATATTGAGGAGACAGAAC IFLKFGSGY ATACAGAGCAAAAGCGAAATGVSGWGRVFH TGATTCGAATTATTCCTCACCAC KGRSALVLQ AACTACAATGCAGCTATTAATAYLRVPLVDR AGTACAACCATGACATTGCCCT ATCLRSTKF TCTGGAACTGGACGAACCCTTATIYNNMFCA GTGCTAAACAGCTACGTTACAC GFHEGGRDS CTATTTGCATTGCTGACAAGGACQGDSGGPH ATACACGAACATCTTCCTCAAA VTEVEGTSF TTTGGATCTGGCTATGTAAGTGLTGIISWGE GCTGGGGAAGAGTCTTCCACAA ECAMKGKYG AGGGAGATCAGCTTTAGTTCTTIYTKVSRYV CAGTACCTTAGAGTTCCACTTG NWIKEKTKL TTGACCGAGCCACATGTCTTCGTGAPLGLRL ATCTACAAAGTTCACCATCTAT RGKLTRAET AACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTC ATGTCAAGGAGATAGTGGGGG ACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA TTATTAGCTGGGGTGAAGAGTG TGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGT ATGTCAACTGGATTAAGGAAAA AACAAAGCTCACTGGGGT0GGT 0FIX- YNSGKLEEF 740 TATAATTCAGGTAAATTGGAAG 741 MMP- VQGNLERECAGTTTGTTCAAGGGAACCTTGA 17- MEEKCSFEE GAGAGAATGTATGGAAGAAAA Elas-AREVFENTE GTGTAGTTTTGAAGAAGCACGA tase RTTEFWKQY GAAGTTTTTGAAAACACTGAAAVDGDQCESN GAACAACTGAATTTTGGAAGCA PCLNGGSCK GTATGTTGATGGAGATCAGTGTDDINSYECW GAGTCCAATCCATGTTTAAATG CPFGFEGKN GCGGCAGTTGCAAGGATGACATCELDVTCNI TAATTCCTATGAATGTTGGTGTC KNGRCEQFC CCTTTGGATTTGAAGGAAAGAAKNSADNKVV CTGTGAATTAGATGTAACATGT CSCTEGYRL AACATTAAGAATGGCAGATGCGAENQKSCEP AGCAGTTTTGTAAAAATAGTGC AVPFPCGRV TGATAACAAGGTGGTTTGCTCCSVSQTSKLT TGTACTGAGGGATATCGACTTG RAETVFPDV CAGAAAACCAGAAGTCCTGTGADYVNSTEAE ACCAGCAGTGCCATTTCCATGT TILDNITQS GGAAGAGTTTCTGTTTCACAAATQSFNDFTR CTTCTAAGCTCACCCGTGCTGA VVGGEDAKP GACTGTTTTTCCTGATGTGGACTGQFPWQVVL ATGTAAATTCTACTGAAGCTGA NGKVDAFCG AACCATTTTGGATAACATCACTGSIVNEKWI CAAAGCACCCAATCATTTAATG VTAAHCVET ACTTCACTCGGGTTGTTGGTGGGVKITVVAG AGAAGATGCCAAACCAGGTCA EHNIEETEH ATTCCCTTGGCAGGTTGTTTTGATEQKRNVIR ATGGTAAAGTTGATGCATTCTG IIPHHNYNA TGGAGGCTCTATCGTTAATGAAAINKYNHDI AAATGGATTGTAACTGCTGCCC ALLELDEPL ACTGTGTTGAAACTGGTGTTAAVLNSYVTPI AATTACAGTTGTCGCAGGTGAA CIADKEYTN CATAATATTGAGGAGACAGAACIFLKFGSGY ATACAGAGCAAAAGCGAAATG VSGWGRVFH TGATTCGAATTATTCCTCACCACKGRSALVLQ AACTACAATGCAGCTATTAATA YLRVPLVDR AGTACAACCATGACATTGCCCTATCLRSTKF TCTGGAACTGGACGAACCCTTA TIYNNMFCA GTGCTAAACAGCTACGTTACACGFHEGGRDS CTATTTGCATTGCTGACAAGGA CQGDSGGPH ATACACGAACATCTTCCTCAAAVTEVEGTSF TTTGGATCTGGCTATGTAAGTG LTGIISWGE GCTGGGGAAGAGTCTTCCACAAECAMKGKYG AGGGAGATCAGCTTTAGTTCTT IYTKVSRYV CAGTACCTTAGAGTTCCACTTGNWIKEKTKL TTGACCGAGCCACATGTCTTCG TGAPLGLRL ATCTACAAAGTTCACCATCTAT RAACAACATGTTCTGTGCTGGCT TCCATGAAGGAGGTAGAGATTC ATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAA GGGACCAGTTTCTTAACTGGAA TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGA ATATATACCAAGGTATCCCGGT ATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTGGGGT0  FVII- ANAFLEELR 742 GCCAACGCGTTCCTGGAGGAGC 743 FIXPGSLERECK TACGGCCGGGCTCCCTGGAGAG AP- EEQCSFEEA GGAGTGCAAGGAGGAGCAGTGAE288 REIFKDAER CTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGADGDQCASSP CGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCTQLQSYICFC CAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAGETHKDDQLI TCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGTQYCSDHTGT GAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACGYSLLADGVS GCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGCGKIPILEKR TCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGTVGGKVCPKG GTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTAVNGAQLCGG TTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTGSAAHCFDKI GGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCCLGEHDLSEH TGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATCAQVIIPSTY AACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAATALLRLHQPV CAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACCCLPERTFSE TCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGCLVSGWGQLL AGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCACMVLNVPRLM GACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGAKVGDSPNIT CCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGADGSKDSCKG GGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGCYRGTWYLTG CAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCTTVGHFGVYT CAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCACQKLMRSEPR GGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTGPGKLTRAET TGCCGGCTACTCGGATGGCAGC VFPDVDYVN AAGGACTCCTGCAAGGGGGACSTEAETILD AGTGGAGGCCCACATGCCACCC NITQSTQSF ACTACCGGGGCACGTGGTACCTNDFTRVVGG GACGGGCATCGTCAGCTGGGGC E CAGGGCTGCGCAACCGTGGGCC GGTSESATPACTTTGGGGTGTACACCAGGGT ESGPGSEPA CTCCCAGTACATCGAGTGGCTG TSGSETPGTCAAAAGCTCATGCGCTCAGAGC SESATPESG CACGCCCAGGAGTCCTCCTGCG PGSEPATSGAGCCCCATTTCCCGGT0GGTGGT SETPGTSES ACCTCTGAAAGCGCAACTCCTG ATPESGPGTAGTCTGGCCCAGGTAGCGAACC STEPSEGSA TGCTACCTCCGGCTCTGAGACT PGSPAGSPTCCAGGTACCTCTGAAAGCGCAA STEEGTSES CCCCGGAATCTGGTCCAGGTAG ATPESGPGSCGAACCTGCAACCTCTGGCTCT EPATSGSET GAAACCCCAGGTACCTCTGAAA PGTSESATPGCGCTACTCCTGAATCTGGCCC ESGPGSPAG AGGTACTTCTACTGAACCGTCC SPTSTEEGSGAGGGCAGCGCACCAGGTAGC PAGSPTSTE CCTGCTGGCTCTCCAACCTCCA EGTSTEPSECCGAAGAAGGTACCTCTGAAAG GSAPGTSES CGCAACCCCTGAATCCGGCCCA ATPESGPGTGGTAGCGAACCGGCAACCTCCG SESATPESG GTTCTGAAACCCCAGGTACTTC PGTSESATPTGAAAGCGCTACTCCTGAGTCC ESGPGSEPA GGCCCAGGTAGCCCGGCTGGCT TSGSETPGSCTCCGACTTCCACCGAGGAAGG EPATSGSET TAGCCCGGCTGGCTCTCCAACT PGSPAGSPTTCTACTGAAGAAGGTACTTCTA STEEGTSTE CCGAACCTTCCGAGGGCAGCGC PSEGSAPGTACCAGGTACTTCTGAAAGCGCT STEPSEGSA ACCCCTGAGTCCGGCCCAGGTA PGSEPATSGCTTCTGAAAGCGCTACTCCTGA SETPGTSES ATCCGGTCCAGGTACTTCTGAA ATPESGPGTAGCGCTACCCCGGAATCTGGCC STEPSEGSA CAGGTAGCGAACCGGCTACTTC PTGGTTCTGAAACCCCAGGTAGC GAACCGGCTACCTCCGGTTCTG AAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA GGTACTTCTACTGAACCTTCCG AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGC GCTCCAGGTAGCGAACCTGCAA CCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGCACCA FVII- ANAFLEELR 744 GCCAACGCGTTCCTGGAGGAGC 745 FIX PGSLERECKTACGGCCGGGCTCCCTGGAGAG AP- EEQCSFEEA GGAGTGCAAGGAGGAGCAGTG AE864REIFKDAER CTCCTTCGAGGAGGCCCGGGAG TKLFWISYS ATCTTCAAGGACGCGGAGAGGADGDQCASSP CGAAGCTGTTCTGGATTTCTTAC CQNGGSCKD AGTGATGGGGACCAGTGTGCCTQLQSYICFC CAAGTCCATGCCAGAATGGGGG LPAFEGRNC CTCCTGCAAGGACCAGCTCCAGETHKDDQLI TCCTATATCTGCTTCTGCCTCCC CVNENGGCE TGCCTTCGAGGGCCGGAACTGTQYCSDHTGT GAGACGCACAAGGATGACCAG KRSCRCHEG CTGATCTGTGTGAACGAGAACGYSLLADGVS GCGGCTGTGAGCAGTACTGCAG CTPTVEYPC TGACCACACGGGCACCAAGCGCGKIPILEKR TCCTGTCGGTGCCACGAGGGGT NASKPQGRI ACTCTCTGCTGGCAGACGGGGTVGGKVCPKG GTCCTGCACACCCACAGTTGAA ECPWQVLLL TATCCATGTGGAAAAATACCTAVNGAQLCGG TTCTAGAAAAAAGAAATGCCAG TLINTIWVV CAAACCCCAAGGCCGAATTGTGSAAHCFDKI GGGGGCAAGGTGTGCCCCAAA KNWRNLIAV GGGGAGTGTCCATGGCAGGTCCLGEHDLSEH TGTTGTTGGTGAATGGAGCTCA DGDEQSRRV GTTGTGTGGGGGGACCCTGATCAQVIIPSTY AACACCATCTGGGTGGTCTCCG VPGTTNHDI CGGCCCACTGTTTCGACAAAATALLRLHQPV CAAGAACTGGAGGAACCTGATC VLTDHVVPL GCGGTGCTGGGCGAGCACGACCCLPERTFSE TCAGCGAGCACGACGGGGATG RTLAFVRFS AGCAGAGCCGGCGGGTGGCGCLVSGWGQLL AGGTCATCATCCCCAGCACGTA DRGATALEL CGTCCCGGGCACCACCAACCACMVLNVPRLM GACATCGCGCTGCTCCGCCTGC TQDCLQQSR ACCAGCCCGTGGTCCTCACTGAKVGDSPNIT CCATGTGGTGCCCCTCTGCCTG EYMFCAGYS CCCGAACGGACGTTCTCTGAGADGSKDSCKG GGACGCTGGCCTTCGTGCGCTT DSGGPHATH CTCATTGGTCAGCGGCTGGGGCYRGTWYLTG CAGCTGCTGGACCGTGGCGCCA IVSWGQGCA CGGCCCTGGAGCTCATGGTCCTTVGHFGVYT CAACGTGCCCCGGCTGATGACC RVSQYIEWL CAGGACTGCCTGCAGCAGTCACQKLMRSEPR GGAAGGTGGGAGACTCCCCAA PGVLLRAPF ATATCACGGAGTACATGTTCTGPGKLTRAET TGCCGGCTACTCGGATGGCAGC VFPDVDYVN AAGGACTCCTGCAAGGGGGACSTEAETILD AGTGGAGGCCCACATGCCACCC NITQSTQSF ACTACCGGGGCACGTGGTACCTNDFTRVVGG GACGGGCATCGTCAGCTGGGGC E CAGGGCTGCGCAACCGTGGGCC GGSPAGSPTACTTTGGGGTGTACACCAGGGT STEEGTSES CTCCCAGTACATCGAGTGGCTG ATPESGPGTCAAAAGCTCATGCGCTCAGAGC STEPSEGSA CACGCCCAGGAGTCCTCCTGCG PGSPAGSPTAGCCCCATTTCCCGGT0GGTGGT STEEGTSTE AGCCCGGCTGGCTCTCCTACCT PSEGSAPGTCTACTGAGGAAGGTACTTCTGA STEPSEGSA AAGCGCTACTCCTGAGTCTGGT PGTSESATPCCAGGTACCTCTACTGAACCGT ESGPGSEPA CCGAAGGTAGCGCTCCAGGTAG TSGSETPGSCCCAGCAGGCTCTCCGACTTCC EPATSGSET ACTGAGGAAGGTACTTCTACTG PGSPAGSPTAACCTTCCGAAGGCAGCGCACC STEEGTSES AGGTACCTCTACTGAACCTTCT ATPESGPGTGAGGGCAGCGCTCCAGGTACTT STEPSEGSA CTGAAAGCGCTACCCCGGAATC PGTSTEPSETGGCCCAGGTAGCGAACCGGCT GSAPGSPAG ACTTCTGGTTCTGAAACCCCAG SPTSTEEGTGTAGCGAACCGGCTACCTCCGG STEPSEGSA TTCTGAAACTCCAGGTAGCCCG PGTSTEPSEGCAGGCTCTCCGACCTCTACTG GSAPGTSES AGGAAGGTACTTCTGAAAGCGC ATPESGPGTAACCCCGGAGTCCGGCCCAGGT STEPSEGSA ACCTCTACCGAACCGTCTGAGG PGTSESATPGCAGCGCACCAGGTACTTCTAC ESGPGSEPA CGAACCGTCCGAGGGTAGCGCA TSGSETPGTCCAGGTAGCCCAGCAGGTTCTC STEPSEGSA CTACCTCCACCGAGGAAGGTAC PGTSTEPSETTCTACCGAACCGTCCGAGGGT GSAPGTSES AGCGCACCAGGTACCTCTACTG ATPESGPGTAACCTTCTGAGGGCAGCGCTCC SESATPESG AGGTACTTCTGAAAGCGCTACC PGSPAGSPTCCGGAGTCCGGTCCAGGTACTT STEEGTSES CTACTGAACCGTCCGAAGGTAG ATPESGPGSCGCACCAGGTACTTCTGAAAGC EPATSGSET GCAACCCCTGAATCCGGTCCAG PGTSESATPGTAGCGAACCGGCTACTTCTGG ESGPGTSTE CTCTGAGACTCCAGGTACTTCT PSEGSAPGTACCGAACCGTCCGAAGGTAGCG STEPSEGSA CACCAGGTACTTCTACTGAACC PGTSTEPSEGTCTGAAGGTAGCGCACCAGGT GSAPGTSTE ACTTCTGAAAGCGCAACCCCGG PSEGSAPGTAATCCGGCCCAGGTACCTCTGA STEPSEGSA AAGCGCAACCCCGGAGTCCGGC PGTSTEPSECCAGGTAGCCCTGCTGGCTCTC GSAPGSPAG CAACCTCCACCGAAGAAGGTAC SPTSTEEGTCTCTGAAAGCGCAACCCCTGAA STEPSEGSA TCCGGCCCAGGTAGCGAACCGG PGTSESATPCAACCTCCGGTTCTGAAACCCC ESGPGSEPA AGGTACCTCTGAAAGCGCTACT TSGSETPGTCCGGAGTCTGGCCCAGGTACCT SESATPESG CTACTGAACCGTCTGAGGGTAG PGSEPATSGCGCTCCAGGTACTTCTACTGAA SETPGTSES CCGTCCGAAGGTAGCGCACCAG ATPESGPGTGTACTTCTACCGAACCGTCCGA STEPSEGSA AGGCAGCGCTCCAGGTACCTCT PGTSESATPACTGAACCTTCCGAGGGCAGCG ESGPGSPAG CTCCAGGTACCTCTACCGAACC SPTSTEEGSTTCTGAAGGTAGCGCACCAGGT PAGSPTSTE ACTTCTACCGAACCGTCCGAGG EGSPAGSPTGTAGCGCACCAGGTAGCCCAGC STEEGTSES AGGTTCTCCTACCTCCACCGAG ATPESGPGTGAAGGTACTTCTACCGAACCGT STEPSEGSA CCGAGGGTAGCGCACCAGGTAC PGTSESATPCTCTGAAAGCGCAACTCCTGAG ESGPGSEPA TCTGGCCCAGGTAGCGAACCTG TSGSETPGTCTACCTCCGGCTCTGAGACTCC SESATPESG AGGTACCTCTGAAAGCGCAACC PGSEPATSGCCGGAATCTGGTCCAGGTAGCG SETPGTSES AACCTGCAACCTCTGGCTCTGA ATPESGPGTAACCCCAGGTACCTCTGAAAGC STEPSEGSA GCTACTCCTGAATCTGGCCCAG PGSPAGSPTGTACTTCTACTGAACCGTCCGA STEEGTSES GGGCAGCGCACCAGGTACTTCT ATPESGPGSGAAAGCGCTACTCCTGAGTCCG EPATSGSET GCCCAGGTAGCCCGGCTGGCTC PGTSESATPTCCGACTTCCACCGAGGAAGGT ESGPGSPAG AGCCCGGCTGGCTCTCCAACTT SPTSTEEGSCTACTGAAGAAGGTAGCCCGGC PAGSPTSTE AGGCTCTCCGACCTCTACTGAG EGTSTEPSEGAAGGTACTTCTGAAAGCGCAA GSAPGTSES CCCCGGAGTCCGGCCCAGGTAC ATPESGPGTCTCTACCGAACCGTCTGAGGGC SESATPESG AGCGCACCAGGTACCTCTGAAA PGTSESATPGCGCAACTCCTGAGTCTGGCCC ESGPGSEPA AGGTAGCGAACCTGCTACCTCC TSGSETPGSGGCTCTGAGACTCCAGGTACCT EPATSGSET CTGAAAGCGCAACCCCGGAATC PGSPAGSPTTGGTCCAGGTAGCGAACCTGCA STEEGTSTE ACCTCTGGCTCTGAAACCCCAG PSEGSAPGTGTACCTCTGAAAGCGCTACTCC STEPSEGSA TGAATCTGGCCCAGGTACTTCT PGSEPATSGACTGAACCGTCCGAGGGCAGCG SETPGTSES CACCAGGTAGCCCTGCTGGCTC ATPESGPGTTCCAACCTCCACCGAAGAAGGT STEPSEGSA ACCTCTGAAAGCGCAACCCCTG PAATCCGGCCCAGGTAGCGAACC GGCAACCTCCGGTTCTGAAACC CCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG CCCGGCTGGCTCTCCGACTTCC ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGGTACTTCTACCGAACCTTCC GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTC CGGCCCAGGTACTTCTGAAAGC GCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC GGAATCTGGCCCAGGTAGCGAA CCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTAC CTCCGGTTCTGAAACTCCAGGT AGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTAC TGAACCTTCCGAAGGCAGCGCA CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAG CGAACCTGCAACCTCTGGCTCT GAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC AGGTACTTCTACTGAACCGTCC GAGGGCAGCGCACCA *Sequencename reflects N- to C-terminus configuration of the CF, cleavagesequence and XTEN components

TABLE 43 Exemplary FVII variants incorporating FIX AP sequence,cleavage sequences and XTEN** FVII SEQ Construct ID Name*Amino Acid Sequence NO: FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 746 EGF2)-(AP)-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG FVII(Pro)CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 747 EGF2)-(AP)-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG FVII(Pro)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA AE288CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPFVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 748EGF2)-(AP)- CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG FVII(Pro)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA AE864SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 749 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V149)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 750 EGF2)-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG (K142-V149)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH AE288CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 751 EGF2)-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG (K142-V149)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH AE864CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 752 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG G184)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 753 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG G184)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH AE288CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 754 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG G184)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH AE864CDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 755 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI EWLQKLMRSEPRPGVLLRAPFPFVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 756EGF2)-(D177- CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH KLTRAET-CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH AE288(‘KLTRAET’DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR disclosed asGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK SEQ ID NO:DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI 6)EWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 757 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH KLTRAET-CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH AE864(‘KLTRAET’DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR disclosed asGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK SEQ ID NO:DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI 6)EWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 758 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL FVII(Pro)TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR APFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 759 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL FVII(Pro)-TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGEC KLTRAET-PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH AE288(‘KLTRAET’DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP disclosed asLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ SEQ ID NO:DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT 6)WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 760 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL FVII(Pro)- TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGEC KLTRAET-PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH AE864(‘KLTRAET’DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP disclosed asLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ SEQ ID NO:DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT 6)WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 761 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL LRAPFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 762 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)-LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGE KLTRAET-CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE AE288(‘KLTRAET’HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV disclosed asVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM SEQ ID NO:TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR 6)GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA P FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 763 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)-LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGE KLTRAET-CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE AE864(‘KLTRAET’HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV disclosed asVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM SEQ ID NO:TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR 6)GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 764 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI EWLQKLMRSEPRPGVLLRAPFPFVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 765EGF2)-(D177- CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH KLTRAET-CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH AE288(‘KLTRAET’DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR disclosed asGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK SEQ ID NO:DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI 6)EWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 766 EGF2)-(D177-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG T179)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA FVII(Pro)-SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH KLTRAET-CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH AE864(‘KLTRAET’DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR disclosed asGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK SEQ ID NO:DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI 6)EWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 767 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL FVII(Pro)TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR APFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 768 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL FVII(Pro)-TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGEC KLTRAET-PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH AE288(‘KLTRAET’DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP disclosed asLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ SEQ ID NO:DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT 6)WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 769 EGF2)-(K142-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL FVII(Pro)-TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGEC KLTRAET-PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH AE864(‘KLTRAET’DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP disclosed asLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ SEQ ID NO:DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT 6)WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 770 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL LRAPFP FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 771 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)-LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGE KLTRAET-CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE AE288(‘KLTRAET’HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV disclosed asVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM SEQ ID NO:TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR 6)GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA P FVII(Gla-ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ 772 EGF2)-(R134-CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG V181)-CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK FVII(Pro)-LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGE KLTRAET-CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE AE864(‘KLTRAET’HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV disclosed asVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM SEQ ID NO:TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR 6)GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS *Sequence name reflects N- to C-terminusconfiguration of the FVII variant (Gla-EGF2 domains), FIX AP cleavagesequence, FVII protease domain, XTEN cleavage sequence and XTENcomponents (the latter when included) **Not all sequences incorporateXTEN

1. An isolated factor IX polypeptide comprising an extended recombinantpolypeptide (XTEN), said XTEN comprising at least 200 amino acidresidues, wherein said factor IX polypeptide exhibits a terminalhalf-life that is longer than about 12 hours when administered to asubject.
 2. The isolated factor IX polypeptide of claim 1, wherein thefactor IX polypeptide exhibits at least 90% sequence identity comparedto a sequence selected from Table 2 when optimally aligned.
 3. Theisolated factor IX polypeptide of claim 1, wherein said factor IX islinked at its C-terminus to the XTEN.
 4. The isolated factor IXpolypeptide of claim 1 that is linked to the XTEN via a cleavagesequence that is cleavable by a mammalian protease selected from thegroup consisting of factor XIa, factor XIIa, kallikrein, factor VIIa,factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13,MMP-17 and MMP-20.
 5. The isolated factor IX polypeptide of claim 4,wherein cleavage at the cleavage sequence by the mammalian proteasereleases said XTEN from said factor IX polypeptide.
 6. The isolatedfactor IX polypeptide of claim 1, wherein said XTEN is characterized inthat: (a) the cumulative total of XTEN amino acid residues is greaterthan 200 to about 3000 amino acid residues; (b) the sum of asparagineand glutamine residues is less than 10% of the total amino acid sequenceof the XTEN; (c) the sum of methionine and tryptophan residues is lessthan 2% of the total amino acid sequence of the XTEN; (d) the XTENsequence has a subsequence score less than 10; (e) the XTEN sequence hasgreater than 90% random coil formation as determined by GOR algorithm;and (f) the XTEN sequence has less than 2% alpha helices and 2%beta-sheets as determined by Chou-Fasman algorithm.
 7. The isolatedfactor IX polypeptide of claim 1 exhibiting an apparent molecular weightfactor of at least about
 4. 8. The isolated factor IX polypeptide ofclaim 1, wherein said XTEN exhibits at least 90% sequence identity to acomparable length of an amino acid sequence selected from Table 4, Table9, Table 10, Table 11, Table 12, or Table
 13. 9. The isolated factor IXpolypeptide of claim 1 that is configured according to formula VII:(Gla)-(XTEN)_(u)-(EGF1)-(XTEN)_(v)-(EGF2)-(AP1)-(XTEN)_(w)-(AP2)-(XTEN)_(x)-(Pro)-(S)_(y)-(XTEN)_(z)  VIIwherein independently for each occurrence, (a) Gla is a Gla domain offactor IX; (b) EGF1 is an EGF1 domain of factor IX; (c) EGF2 is an EFG2domain of factor IX; (d) AP1 is a portion of an activator peptide domainof factor IX; (e) AP2 is a portion of an activator peptide domain offactor IX that includes at least a first cleavage sequence; (f) Pro is aprotease domain of factor IX; (g) S is a spacer sequence having between1 to about 50 amino acid residues that can optionally include a cleavagesequence; (h) XTEN is an extended recombinant polypeptide that exhibitsat least 90% sequence identity to a comparable length of an amino acidsequence selected from Table 4, Table 9, Table 10, Table 11, Table 12,or Table 13; (i) u is either 0 or 1; (j) v is either 0 or 1; (k) w iseither 0 or 1; (l) x is either 0 or 1; (m) y is either 0 or 1; and (n) zis either 0 or 1, with the proviso that u+v+x+y+z≥1.
 10. The isolatedfactor IX polypeptide of claim 9, wherein y=1 and S comprises a cleavagesequence that is cleavable by a mammalian protease selected from thegroup consisting of FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa(thrombin), Elastase-2, MMP-12, MMP-13, MMP-17 and MMP-20.
 11. Theisolated factor IX polypeptide of claim 1 comprising more than one XTEN.12. The isolated factor IX polypeptide of claim 1, wherein said XTEN isincorporated between any two adjacent domains contained in said factorIX, wherein said two adjacent domains are selected from the groupconsisting of Gla, EGF1, EGF2, AP, and peptidase S1 (Pro).
 13. Theisolated factor IX polypeptide of claim 1, characterized in that: (i) ithas a longer terminal half-life when administered to a mammal comparedto the corresponding factor IX that lacks the XTEN when administered toa mammal at a comparable molar dose; (ii) when a smaller molar amount ofthe factor IX polypeptide is administered to a mammal in comparison tothe corresponding factor IX that lacks the XTEN administered to a mammalunder an otherwise equivalent dose regimen, the factor IX polypeptideachieves a comparable area under the curve (AUC) as the correspondingfactor IX that lacks the XTEN; (iii) when a smaller molar amount of thefactor IX polypeptide is administered to a mammal in comparison to thecorresponding factor IX that lacks the XTEN administered to a mammalunder an otherwise equivalent dose regimen, the factor IX polypeptideachieves a comparable therapeutic effect as the corresponding factor IXthat lacks the XTEN; (iv) when the factor IX polypeptide is administeredto a mammal less frequently in comparison to the corresponding factor IXthat lacks the XTEN administered to a mammal using an otherwiseequivalent molar amount, the factor IX polypeptide achieves a comparablearea under the curve (AUC) as the corresponding factor IX that lacks theXTEN; (v) when the factor IX polypeptide is administered to a mammalless frequently in comparison to the corresponding factor IX that lacksthe XTEN administered to a mammal using an otherwise equivalent molaramount, the factor IX polypeptide achieves a comparable therapeuticeffect as the corresponding factor IX that lacks the XTEN; (vi) when anaccumulatively smaller molar amount of the factor IX polypeptide isadministered to a mammal in comparison to the corresponding factor IXthat lacks the XTEN administered to a mammal under an otherwiseequivalent dose period, the factor IX polypeptide achieves comparablearea under the curve (AUC) as the corresponding factor IX that lacks theXTEN; or (vii) when an accumulatively smaller molar amount of the factorIX polypeptide is administered to a mammal in comparison to thecorresponding factor IX that lacks the XTEN administered to a mammalunder an otherwise equivalent dose period, the factor IX polypeptideachieves comparable therapeutic effect as the corresponding factor IXthat lacks the XTEN.
 14. A method of treating coagulopathy in a subject,comprising administering to said subject a composition comprising atherapeutically effective amount of the factor IX polypeptide ofclaim
 1. 15. The method of claim 14, wherein said coagulopathy ishemophilia B.
 16. A method of treating a bleeding episode in a subjectcomprising administering to said subject a composition comprising atherapeutically effective amount of the factor IX polypeptide ofclaim
 1. 17. A method of treating a subject deficient in a clottingprotein, comprising: administering to said subject a compositioncomprising a therapeutically effective amount of the factor IXpolypeptide of claim
 1. 18. The method of claim 17, wherein the factorIX polypeptide substitutes wild-type factor VII, factor IX, or factorXI.